Ganglioside-KLH conjugate vaccine with QS-21

ABSTRACT

This invention provides a vaccine for stimulating or enhancing in a subject to which the vaccine is administered, production of an antibody which recognizes a ganglioside, comprising an amount of ganglioside or oligosaccharide portion thereof conjugated to an immunogenic protein effective to stimulate or enhance antibody production in the subject, an effective amount of adjuvant and a pharmaceutically acceptable vehicle.

This invention was made with support under Government Grant No. R01CA40532. Accordingly, the U.S. Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Gangliosides are sialic acid containing glycosphingolipids composed of acomplex carbohydrate moiety linked to a hydrophobic ceramide portion.Embedded within the outer leaflet of the cell membrane, the carbohydratechain is exposed to the extracellular matrix. Qualitative andquantitative changes in ganglioside composition during celldifferentiation and proliferation have been observed and seem to reflectthe state of malignant transformation of cancers of neuroectodermalorigin (Hakomori 1985). Malignant melanoma cells express a variety ofcomplex gangliosides in addition to GM3, the major ganglioside in normalmelanoctyes (Carubia et al. 1984). Altered ganglioside metabolism inmelanoma causes additional expression of GD3, GD2, GM2, 9-O-Acetyl-GD3and GT3 (Hamilton et al. 1993; Tsuchida et al., 1987). Treatment ofpatients with anti-GD3 monoclonal antibodies resulted in inflammation atthe tumor site and partial regression of metastasis was seenoccasionally, suggesting, that gangliosides are suitable targets forimmune attack (Houghton et al., 1985). The generation of human MAb'sreactive with GD3 from melanoma patients (Yamaguchi et al., 1987)support the idea, that gangliosides are potential immunogens as well.

In studies aimed at inducing a humoral response against gangliosides inmelanoma patients by active immunization, GM2/BCG vaccines seemed to bemost effective (Livingston et al., 1987; Livingston et al., 1989). In arandomized study with 122 melanoma patients, who were disease-free aftersurgery, that it was showed that, out of 64 patients treated with BCGalone and 58 patients with GM2/BCG, the majority of patients (86%)receiving the GM2 vaccine produced antibodies. Patients that producedanti-GM2 antibodies had a significantly longer disease free and overallsurvival than antibody negative patients. Comparing the two arms of thetrial, patients receiving the GM2/BCG vaccine had a 17% improvement indisease-free interval and 9% improvement in survival when compared tothe BCG control group, though neither result was statisticallysignificant (Livingston et al., 1993a). Unfortunately, the immuneresponse was only of short duration, mostly IgM and of moderate titer.This suggested that GM2 was recognized as a T-cell independent antigenas a consequence of carbohydrate antigens (Livingston et al., 1989) andalso because gangliosides are auto antigens expressed on some normaltissue (Hamilton et al., 1993). Similar approaches with GD2 and9-O-Acetyl-GD3 vaccines in patients resulted in occasionally low titersand no antibody response against GD3 could be detected (Livingston,1991).

New potent adjuvants were able to enhance the immune responses againstgangliosides in some cases, but especially for auto antigens such as fortumor associated gangliosides a different approach had to be utilized.Based on Landsteiner's classical experiments (Landsteiner and Chase,1942) with hapten-carrier conjugates, covalent attachment of poorlyimmunogenic antigens to immunogenic carrier proteins has beensuccessfully used to enhance immune response. For example responsivenessto carbohydrates, other than gangliosides, could be accomplished withconjugation to appropriate carrier proteins. Coupling of bacterialcapsular polysaccharides to immunogenic proteins showed a significantincrease in immune response and protection (Eskola et al., 1990).Recently, vaccination of ovarian cancer patients with synthetic ThompsonFriedenreich tumor antigen conjugated to keyhole limpet hemocyaninelicited humoral IgM and IgG response (MacLean et al., 1992). Theimportant finding common in these studies was the isotype switch from aIgM response of short duration to a long lasting, high affinity IgGresponse indicating that activation of T-cell dependent pathways againstcarbohydrates is likely to occur. This approach is now applied to themelanoma tumor antigen GD3 to develop a method to synthesizeganglioside-protein conjugate vaccines and examine the immunogenicity ofdifferent GD3-protein conjugates in mice.

SUMMARY OF THE INVENTION

This invention provides a vaccine for stimulating or enhancing in asubject to which the vaccine is administered, production of an antibodywhich recognizes a ganglioside, comprising an amount of ganglioside oroligosaccharide portion thereof conjugated to an immunogenic proteineffective to stimulate or enhance antibody production in the subject, aneffective amount of adjuvant and a pharmaceutically acceptable vehicle.

This invention also provides a method for stimulating or enhancing in asubject production of antibodies which recognize a gangliosidecomprising administering to the subject an effective dose of a vaccinefor stimulating or enhancing in a subject to which the vaccine isadministered, production of an antibody which recognizes a ganglioside,comprising an amount of ganglioside or oligosaccharide portion thereofconjugated to an immunogenic protein effective to stimulate or enhanceantibody production in the subject, an effective amount of adjuvant anda pharmaceutically acceptable vehicle.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 The Synthesis of GD3 protein conjugates after ozone cleavage andreductive amination. Insert represents HPTLC of GD3 before (A) and after(B) the cleavage.

FIGS. 2A and 2B Time course of GD3-KLH antisera IgM (a) and IgG (b)antibodies. Each symbol on the figure represents a mouse.

FIGS. 3A and 3B Immune thin layer chromatography of three mouse antiseraafter vaccination with GD3-KLH. Reactivities of IgG (a) and IgM (b)antibodies were tested on A, human brain gangliosides, B neuroblastomagangliosides, C, melanoma ganglioside and D, GD3 antigen.

FIG. 4 Immunoblot of four different mice to show the specificity of theimmune response. Pure ganglioside are dot-blotted and incubated withsera from mice.

FIG. 5 Representative FACS analysis of mouse serum reactivity prior to(peak at 3) and after (peak at 50) immunization with G_(D3)-KLH andQS-21 tested on melanoma cell line SK-MEL-28

FIGS. 6A and 6B Time course of GM2-KLH antisera IgM (a) and IgG (b)antibodies. Each symbol on the figure represents a patient.

FIG. 7 Detection of GM2 antibody in sera from patients vaccinated withGM2 conjugate vaccine plus adjuvant by dot blot immune staining.Ganglioside standards were spotted on nitrocellulose strips (indicatedon the vertical axis) and allowed to react with prevaccination and peaktiter postvaccination sera from individual patients andperoxidase-labeled goat anti-human IgM or IgG antibody. Strips aregraded on a scale from 0 to 3+. MAb 696 was used as positive control forGM2.

FIGS. 8A-1 and 8A-2 Specificity of peak titer sera from patientsimmunized GM2-KLH+QS-21 vaccine determined by immune thin layerchromatography as described previously (3, Reference of the Third Seriesof Experiments). GM2 (A) and melanoma tissue ganglioside extract (B)were applied to TLC plates, incubated with sera from individual patientsand stained with peroxidase-labeled goat anti-human IgM or IgG antibody.MAb 696 was used as positive control for GM2 and resorcinol stain forgangliosides.

FIG. 8B Inhibition of IgG reactivity of patient serum against GM2 andGD2. GM2 (A) and melanoma tissue ganglioside extract (B) were applied toHPTLC plates, incubated with serum from patient No. 2 and stained withperoxidase-labeled goat anti-human IgG antibody. 3 ml Patient serum at adilution of 1:50 was preincubated with either 150 μg GM2 or 150 μg GD2prior to immune staining.

FIGS. 9A and 9B IgM and IgG antibody responses in melanoma patientsafter immunization with GM2-KLH plus QS-21 vaccines. Sequential resultsfor six patients receiving the 100 ug QS-21 dose are shown in FIG. 9 aand for six patients receiving the 200 ug dose in FIG. 9 b. Note thatone patient in each group received only four vaccinations and was takenoff study due to disease progression. Arrows indicate the time ofcyclophosphamide (Cy) and GM2-KLH plus QS-21 vaccine injections.

FIGS. 10A and 10B Detection of GM2 antibody by dot blot immune stainingwith sera from ten patients vaccinated with GM2-KLH. Gangliosidestandards were applied to nitrocellulose strips (as indicated on theleft) and incubated first with sera and then, after washing, withperoxidase labelled goat anti-human IgM or IgG antibody. Results withsera from two patients from each of the five groups receiving differentQS-21 doses (as indicated at the top) are shown. Pre- (a) andpost-immunization (b) sera are shown for each patient. Murine monoclonalantibodies 696 and 3F8 are IgM and IgG antibodies (respectively) againstGM2 and GD2. IgM antibody against GM1 was detected in sera from mostpatients before and after vaccination. IgM and IgG antibody against GM2was not detected before vaccination in any of these patients. Aftervaccination IgM and IgG antibodies were detected against GM2 in serafrom all patients. Reactions were graded 0, 1+, 2+ or 3+. An examplereaction grading for this assay is: Patient 1 (100 ug QS-21) IgM(pre/post vaccination): KLH 1+/2+, GM3 0/0, GM2 0/3+, GM1 1+/1+, GD30/0, GD2 0/1+, GD1b 0/0.

FIGS. 11A and 11B IgM antibody responses in melanoma patients afterimmunization with the GM2/BCG vaccine. Sequential results for fivepatients treated during the initial four months of the protocol (groupA) and five patients treated during the final four months of theprotocol (group B) are shown. Arrows indicate time of cyclophosphamide(Cy) and GM2/BCG vaccine injections.

FIG. 12 Detection of GM2 antibody by dot blot immune staining in serafrom ten GM2/BCG vaccinated melanoma patients. Ganglioside standardswere applied to nitrocellulose strips (as indicated on the left) andincubated first with sera and then after washing incubated withperoxidase labelled goat anti-human IgM antibody. GM2-MEL indicatespurified GM2 extracted from melanoma biopsy samples, all othergangliosides including GM2 were derived from bovine brain. Patientnumbers (6 to 58) are indicated, and pre- (a) and post- (b) immunizationsera are shown for each patient. 696 and 3G6 are IgM murine monoclonalantibodies against GM2 and GD2 respectively. GM2 antibody was detectedin post vaccination sera from these 10 patients. GM1 antibody was seenin pre- and post-treatment sera from patient 53 and in the posttreatment serum from patient 57. Reactions were graded 0, 1+, 2+, or 3+.Examples of reaction gradings are as follows: patient 8: GM2 3+, GM2-MEL2+, patient 54: GM2 3+, GM2-MEL 1+ and patient 57: GM2 3+, GM2-MEL 3+.The lower reactivity against GM2-MEL compared to GM2 seen with postvaccine sera and murine monoclonal antibody 696 reflects a lowerquantity of GM2-MEL ganglioside applied to the strips.

FIG. 13 Kaplan-Meier plots of disease-free and overall survival ofpatients with post vaccination GM2 antibody production. Patients werecategorized as positive if GM2 reactivity was a) 2+ or 3+ by dot blotwith an ELISA titer ≧1/20 or b) 1+ by dot blot with an ELISA titer≧1/80.

FIG. 14 Kaplan-Meier plots of disease-free and overall survival of 59patients randomized to receive BCG and 57 patients randomized to receiveGM2/BCG, excluding six patients who produced GM2 antibody prior toimmunization (five in the BCG arm and one in the GM2/BCG arm).

FIG. 15 Kaplan-Meier plots of disease-free and overall survival of 64patients randomized to receive BCG and 58 patients randomized to receiveGM2/BCG.

FIG. 16 Kaplan-Meier plots of disease-free survival of 59 GM2antibody-negative patients randomized to receive BCG (Δ or ▴) comparedto 57 GM2 antibody-negative patients randomized to receive GM2/BCG (□ or▪). Patients are stratified into two groups, patients with a singlepositive lymph node (Δ or □) and patients with two or more positivelymph nodes (▴ or ▪).

DETAILED DESCRIPTION OF THE INVENTION

Throughout this application, various references are referred to withinparentheses. Disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains. Fullbibliographic citation for these references may be found at the end ofthis application, preceding the claims.

This invention provides a vaccine for stimulating or enhancing in asubject to which the vaccine is administered, production of an antibodywhich recognizes a ganglioside, comprising an amount of ganglioside oroligosaccharide portion thereof conjugated to an immunogenic proteineffective to stimulate or enhance antibody production in the subject, aneffective amount of adjuvant and a pharmaceutically acceptable vehicle.

The oligosaccharide portion of a ganglioside may be derived by cleavinga ganglioside or it may be synthesized directly. As used herein, animmunogenic protein is a protein that, when conjugated to theganglioside or oligosaccharide portion thereof, stimulates or enhancesantibody production in the subject.

In an embodiment of this invention, the subject is a human.

This invention also provides the above-described vaccine wherein theganglioside or oligosaccharide portion thereof is conjugated to KeyholeLimpet Hemocyanin or a derivative of Keyhole Limpet Hemocyanin.

Keyhole Limpet Hemocyanin is a well-known protein. A derivative ofKeyhole Limpet Hemocyanin may be generated by direct linkage of at leastone immunological adjuvant such as monophospholipid A or non-ionic blockcopolymers or cytokine with Keyhole Limpet Hemocyanin. Cytokines arewell known to an ordinary skilled practitioner. An example of cytokineis interleukin 2. There are other known interleukins in the art whichmay be linked to Keyhole Limpet Hemocyanin, forming a derivative ofKeyhole Limpet Hemocyanin.

In an embodiment of the above-described vaccine the adjuvant is QS-21.

There are other known adjuvants which may be applicable to thisinvention. There may be classes of QS-21 or QS-21 like chemicals whichmay be similarly used in accordance with this invention.

This invention further provides the above-described vaccine wherein theganglioside is selected from the group consisting of GM2, GM3, GD2, GD3,GD3 lactone, O-Acetyl GD3 and GT3.

In one of the preferred embodiments of this invention, the gangliosideis GM2. In another embodiment, the ganglioside is GD3. In anotherembodiment, the ganglioside is GD2.

Different effective amounts of the conjugated ganglioside oroligosaccharide portion thereof may be used according to this invention.A person of ordinary skill in the art can perform simple titrationexperiments to determine what the effective amount is required foreffective immunization. An example of such titration experiment is toinject different amounts of the conjugated ganglioside or conjugatedoligosaccharide portion thereof to the subject and then examine theimmune response.

In an embodiment, the effective amount of conjugated ganglioside orconjugated oligosaccharide portion thereof is an amount between about 1μg and about 200 μg.

In another embodiment, the effective amount of conjugated ganglioside orconjugated oligosaccharide portion thereof is an amount between about 50μg and about 90 μg. In an embodiment, the effective amount of conjugatedganglioside or conjugated oligosaccharide portion thereof is about 70μg.

In another embodiment, the effective amount of conjugated ganglioside orconjugated oligosaccharide portion thereof is between about 1 μg andabout 10 μg. In a more specific embodiment, the effective amount ofconjugated ganglioside or conjugated oligosaccharide portion thereof isbetween about 7 μg and about 10 μg. In an embodiment, the effectiveamount of conjugated ganglioside or conjugated oligosaccharide portionthereof is about 7 μg.

In addition, the effective amount of the adjuvant may also be similarlydetermined i.e. administering different amount of the adjuvant with theconjugates and examining the immune response so as to determine whichamount is effective. When using QS-21 as adjuvant, the effective amountof QS-21 may also be similarly determined.

In a preferred embodiment, the effective amount of QS-21 is an amountbetween about 10 μg and about 200 μg. In an embodiment, the effectiveamount of QS-21 is about 100 μg. In another embodiment, the effectiveamount of QS-21 is about 200 μg.

This invention further provides a vaccine for stimulating or enhancingin a subject to which the vaccine is administered, production of anantibody which recognizes a ganglioside, comprising an amount ofganglioside or oligosaccharide portion thereof conjugated to animmunogenic protein effective to stimulate or enhance antibodyproduction in the subject, an effective amount of adjuvant and apharmaceutically acceptable vehicle, wherein the subject is afflictedwith cancer and the antibody produced in the subject-upon administrationof the vaccine effectively treats the cancer.

This invention also provides a vaccine for stimulating or enhancing in asubject to which the vaccine is administered, production of an antibodywhich recognizes a ganglioside, comprising an amount of ganglioside oroligosaccharide portion thereof conjugated to an immunogenic proteineffective to stimulate or enhance antibody production in the subject, aneffective amount of adjuvant and a pharmaceutically acceptable vehicle,wherein the subject is susceptible to cancer and the antibody producedin the subject upon administration of the vaccine effectively preventsthe cancer.

This invention further provides a vaccine for cancers, wherein cells ofthe cancer have gangliosides on their surface.

This invention also provides a vaccine for cancers, wherein gangliosidesare found in the stroma of the of the cancer.

This invention provides a vaccine for cancers which is of epithelial,mesodermal or neuroectodermal origin. Examples of epithelial cancers arebreast cancers and endometrial cancers of the uterus. An example of amesodermal origin cancer is sarcoma. One example of a neuroectodermalorigin cancer is a melanoma.

This invention also provides a method for stimulating or enhancing in asubject production of antibodies which recognize a gangliosidecomprising administering to the subject an effective dose of a vaccinefor stimulating or enhancing in a subject to which the vaccine isadministered, production of an antibody which recognizes a ganglioside,comprising an amount of ganglioside or oligosaccharide portion thereofconjugated to an immunogenic protein effective to stimulate or enhanceantibody production in the subject, an effective amount of adjuvant anda pharmaceutically acceptable vehicle.

In an embodiment of the above-described method, the ganglioside is GM2.

This invention further provides a method for treating cancer in asubject afflicted with cancer comprising administering to the subject aneffective dose of a vaccine for stimulating or enhancing in a subject towhich the vaccine is administered, production of an antibody whichrecognizes a ganglioside, comprising an amount of ganglioside oroligosaccharide portion thereof conjugated to an immunogenic proteineffective to stimulate or enhance antibody production in the subject, aneffective amount of adjuvant and a pharmaceutically acceptable vehicleswherein the subject is afflicted with cancer and the antibody producedin the subject upon administration of the vaccine effectively treats thecancer.

This invention further provides a method for preventing cancer in asubject susceptible to cancer comprising administering to the subject aneffective dose of a vaccine for stimulating or enhancing in a subject towhich the vaccine is administered, production of an antibody whichrecognizes a ganglioside, comprising an amount of ganglioside oroligosaccharide portion thereof conjugated to an immunogenic proteineffective to stimulate or enhance antibody production in the subject, aneffective amount of adjuvant and a pharmaceutically acceptable vehicle,wherein the subject is susceptible to cancer and the antibody producedin the subject upon administration of the vaccine effectively preventsthe cancer.

This invention also provides a method of using the above-describedvaccine, wherein the ganglioside or oligosaccharide portion thereof isconjugated to Keyhole Limpet Hemocyanin or a derivative of KeyholeLimpet Hemocyanin. This invention further provides a method of using theabove-described vaccine wherein the adjuvant is QS-21.

This invention further provides a method of using the above-describedvaccine for treating or preventing cancer, wherein cells of the cancerhave gangliosides on their surface.

This invention further provides a method of using the above-describedvaccine for treating or preventing cancer, wherein gangliosides arefound in the stroma of the cancer.

This invention further provides a method of using the above-describedvaccine for treating or preventing cancer, wherein the cancer is ofepithelial origin or neuroectodermal origin. One such cancer ofneuroectodermal origin is a melanoma.

For the purposes of this invention “pharmaceutically acceptablevehicles” means any of the standard pharmaceutical vehicles. Examples ofsuitable vehicles are well known in the art and may include, but notlimited to, any of the standard pharmaceutical vehicles such as aphosphate buffered saline solutions, phosphate buffered salinecontaining Polysorb 80, water, emulsions such as oil/water emulsion, andvarious type of wetting agents.

The vaccine of this invention may be administered intradermally,subcutaneously and intramuscularly. Other methods well known by a personof ordinary skill in the art may also be used.

In a preferred embodiment this invention provides a method forstimulating or enhancing in a subject production of antibodies whichrecognize a ganglioside comprising administering to the subject aneffective dose of a vaccine for stimulating or enhancing in a subject towhich the vaccine is administered, production of an antibody whichrecognizes a ganglioside, comprising an amount of ganglioside oroligosaccharide portion thereof conjugated to an immunogenic proteineffective to stimulate or enhance antibody production in the subject, aneffective amount of adjuvant and a pharmaceutically acceptable vehicle,wherein the administering comprises administering the effective dose attwo or more sites. “Administering the effective dose at two or moresites” means that the effective dose is divided into two or moreportions and each portion is administered at a different site of thesubject. In a specific embodiment, the administering comprisesadministering at three sites.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

EXPERIMENTAL DETAILS First Series of Experiments

Experimental Details

Increased immunogenicity of GD3 conjugate vaccines: Comparison ofvarious carrier proteins and selection of GD3-KLH for further testing.

Tumor associated gangliosides are known to be suitable targets forimmune attack against cancer but they are poorly immunogenic. Activeimmunization results in low titer antibody IgM responses of shortduration. Covalent attachment of poorly immunogenic antigens toimmunogenic carrier proteins is a potent method for enhancing thehumoral response. GD3, a dominant ganglioside on malignant melanoma, wasattached to carrier proteins by two methods. It was bound by the glucoseof GD3 oligosaccharide but this resulted in loss of antigenicity andinduction of antibodies that failed to react with GD3 or GD3 expressingmelanoma cells. In the second method GD3 was modified by ozone cleavageof the double bond in the ceramide backbone, an aldehyde group wasintroduced and this group was coupled by reductive amination toaminolysyl groups of proteins. Utilizing this method, conjugates wereconstructed with synthetic multiple antigenic peptides (MAP) expressingrepeats of a malaria T-cell epitope, outer membrane proteins (OMP) ofNeisseria meningitidis, cationized bovine serum albumin (cBSA), keyholelimpet hemocyanin (KLH) and polylysine. The antigenicity of conjugateswas confirmed by reactivity with various antibodies and theimmunogenicity was tested in mice. Antibody levels in immune sera wereanalyzed by ELISA and by dot blot immune stains on purifiedgangliosides. Specificity of sera reactivity was further analyzed byimmune thin layer chromatography using tumor tissue extracts. GD3conjugate vaccines resulted in significantly improved antibodyresponses, especially with GD3-KLH conjugates. High titer IgM and IgGresponses against GD3 were induced. This method is applicable to othergangliosides and may be suitable for construction of gangliosidevaccines against a variety of ganglioside rich human cancers.

Materials and Methods

Glycolipids. GM3, GM2 and GD1b and extracted from bovine brain, wereprovided by Fidia Research Laboratory (Abano Terme, Italy). GD2 was madefrom GD1b by treatment with β-galactosidase (Cahan et al., 1982). GD3(mel) was isolated from human melanoma tissue (Ritter et al., 1991), GD3(bbm) (used for vaccine preparation) and GT3 were isolated from bovinebuttermilk and kindly provided by Dr. R. K. Yu (Medical College ofVirginia, Richmond, Va.)(Ritter et al., 1990a). Disialyllactose (GD3oligosaccharide) was isolated from bovine colostrum as previouslydescribed (Nicolai et al., 1978).

Chemicals. HPTLC silica gel plates were obtained from E. Merck(Darmstadt, FRG); Sep-Pak C18 cartridges from Walters Associates(Mildford, Mass.); 4-chloro-1naphtol, p-nitrophenyl phosphate disodium,sodium cyanoborohydride from Sigma Chemical Co. (St. Louis, Mo.);cyclophosphamide (Cytoxan) from Mead Johnson (Syracuse, N.Y.); QS-21containing a saponin Quil A component from Cambridge Biotech (Worcester,Mass.).

Proteins. Poly-L-lysine hydrobromide (MW(vis)3800) was purchased fromSigma; Keyhole limpet hemocyanin (KLH) from Calbiochem (LaJolla,Calif.); cBSA-Imject Supercarrier Immunemodulator from Pierce (Rockfort,Ill.); Neisseria meningitidis outer membrane proteins (OMP) were kindlyprovided by Dr. M. S. Blake (Rockefeller University, New York, N.Y.).Multiple Antigenic Peptide (MAP) YAL-IV 294-I containing 4 repeat of amalarial T-cell epitope was a gift from Dr. J. P. Tam (RockefellerUniversity, New York, N.Y.).

Monoclonal Antibodies. Rabbit anti-mouse immunoglobulins conjugated tohorseradish peroxidase for ITLC, and rabbit anti-mouse IgM and IgGconjugated to alkaline phosphatase for ELISA, were obtained from Zymed(San Francisco, Calif.); anti-GD3 mAb R24 was generated (Houghton etal., 1985).

Serological Assays. Enzyme-linked Immunosorbent Assays (ELISA) wereperformed as previously described (Livingston et al., 1989). To controlfor nonspecific “stickiness”, immune sera were also tested on plateswhich were processed identically but to which no ganglioside had beenadded, and the reading was subtracted from the value obtained in thepresence of ganglioside. The titer was defined as the highest dilutionyielding a corrected absorbance of 0.1 or greater. Immunostaining ofgangliosides with monoclonal antibodies or mouse sera was performedafter separation on high performance thin layer chromatography (HPTLC)silica gel glass plates as previously described (Hamilton et al., 1993).Plates were developed in solvent 1: chloroform/methanol/water (0.25%CaCl₂) 50:40:10 (v/v) or solvent 2:ethanol/n-butanol/pyridin/water/acetic acid 100:10:10:30:3 (v/v) andwere visualized with resorcinol/HCl reagent as well.

Immunization. Six-week-old female BALB/c×C57BL76 F1 mice (The JacksonLaboratory, Bar Harbor, Me.) were given i.p. injections ofcyclophosphamide (15 mg/kg) 3 days before the first immunization andrandomly assigned to treatment groups. Groups of 4 or 5 mice were givens.c. injections of a give three vaccines 2 weeks apart if not otherwiseindicated. Each vaccine contained 20 ug GD3 or 15 ug Disialyllactoseplus 10 ug QS-21 in a total volume of 0.1 ml PBS/mouse. Mice were bledfrom the retro-orbital sinus before and 2 weeks after the vaccine if nototherwise indicated.

GD3 conjugate preparation. GD3 (2 mg) was dissolved in 2 ml methanol bysonication and cooled to −78C in an ethanol/dry-ice bath. Ozone wasgenerated in a ozone generator (Del Industries, San Luis Obispo, Calif.)and was conducted through the sample for 30 minutes under vigorousstirring (Criegee, 1957; Wiegandt and Baschang, 1965). Excess of ozonewas displaced with nitrogen during 10 minutes. 100 ul S(CH₃)₂ was added(Pappas et al., 1966), the sample kept at −78° C. for 30 min, then atroom temperature for 90 min under vigorous stirring. The sample wasdried under a stream of nitrogen and monitored by HPTLC. The long chainaldehyde was separated by adding 2 ml n-hexane to the dry sample,followed by sonication for 5 min and centrifugation at 2000 g for 15min. The n-hexane was carefully drawn off and discarded, and the samplewas dried under a stream of nitrogen. Cleaved GD3 and native GD3 wereseparated by HPLC (Waters, System 501, Milford, Mass.) utilizing a C18reversed phase column (10×250 mm, Rainin Instruments, Ridgefield, N.J.).Gangliosides were eluted with methanol, monitored at 214 nm andfractions were analyzed by HPTLC as well. Fractions that containedcleaved GD3 were combined and were evaporated at 37° C. with a rotavapor(Buchi, Switzerland). Cleaved GD3, protein carrier in PBS and 2 mgsodium cyanoborohydride were incubated under gentle agitation at 37° C.for 48 h. After 16 h another 1 mg NaCNBH₃ was added. The progress ofcoupling was monitored on HPTLC. In solvent 1 and solvent 2 GD3-proteinconjugates did not migrate and appeared as a resorcinol positive band atthe origin. The mixture was dialyzed across 1000 MWCO dialysis tubingwith three changes of each 41 of PBS at 4° C. for 48 h and were passedthrough an Extractigel detergent removing gel (Pierce) for finalpurification of unconjugated GD3. The samples were lyophilized and theirprotein and ganglioside content was determined by Biorad protein assayand by neuraminic acid determination according to Svennerholm (1957).

Disialyllactose was isolated from bovine colostrum as describedpreviously (Nicolai et al., 1978). The carbohydrate was attached toprotein by reductive amination (Gray, 1974) 10 mg disialyllacotse wasincubated with 2 mg of proteins in 2 ml PBS for 14 days at 37° C. 2 mgsodium cyanoborohydride was added at the beginning and 1 mg was addedadditional every 3 days. The coupling was monitored by HPTLC in solvent2. The disialyllactose conjugates were purified by dialysis across 1000MWCO dialysis membrane followed by lyophilization. The protein andneuraminic acid content was determined as described above.Disialyllactose was also conjugated to proteins according to a methoddescribed by Roy and Laferriere (1990). During this procedureN-acroloyled glycopyranosylamine derivatives of the oligosaccharide wereformed first, followed by conjugation via Michael addition to aminogroups of the protein. Purification and protein and neuraminic aciddetermination was performed as described above.

Determination of IgG subclass. The determination of IgG subclass wasperformed by ELISA using subclass-specific secondary MAbs. SecondaryMabs were used at lowest dilution that did not show reactivity withpresera or negative control sera. Alkaline phosphatase conjugated togoat anti-mouse was used as third antibody at a dilution of 1:200.

FACS Analysis of Mouse Antisera

A single cell suspension of the melanoma cell line SK-MEL-28 wasobtained after treatment with 0.1% EDTA in PBS followed by passagethrough a 26½ gauge needle. Cells (3×10⁵) were incubated with 40 μl of1:20 diluted post- or pre-immunization serum for 30 minutes on ice. Thecells were washed three times with 3% fetal calf serum in PBS. Thirty μlof diluted (1:50) fluorescein isothiocyanate-labeled goat anti-mouse IgG(Southern Biotechnology Associates Inc., Birmingham, Ala.) were added assecondary antibody, followed by incubation ice for 30 min. Cells werewashed three times as above and resuspended in 500 μl 3% fetal calfserum in PBS and analyzed by flow cytometry (FACScan, Becton Dickinson,San Jose, Calif.).

Results

Preparation and Characterization of GD3 Vaccines

GD3 from bovine buttermilk was selectively cleaved at the C₄-C₅ doublebond in the ceramide portion by using ozone. In methanol,methoxyperoxides appear to be intermediate products which are readilyreduced with dimethylsulfite. The result of this cleavage was a GD3derivative with an aldehyde functional group at the position of theformer double bond in the ceramide portion and the elimination of a longchain aldehyde (FIG. 1). Successfully cleaved GD3 migrated below nativeGD3, and due to simultaneously cleaved unsaturated fatty acids itappeared as a double band on HPTLC (see HPTLC, insert in FIG. 1).Densitometric determination of HPTLC revealed a cleavage of >70% of GD3isolated from bovine buttermilk. Initial experiments with prolongedozone treatment periods did not change the ratio, indicating that −30%of GD3 from this source consist of sphinganin or phytosphingosineanalogs. Cleavage of GD3 at −78° C. with a reaction time of up to 1 hdepending on the amount of GD3 used, was found to be optimal. CleavedGD3 persisted only in acidic and neutral phosphate buffers for up to 72h but with increasing amount of a byproduct. Due to B-eliminationreactions, release of the oligosaccharide part of GD3 occurredincreasingly with time as has been described earlier to take placereadily at a basic pH (Wiegandt and Baschang, 1965). The carbohydratepart released from GD3 did not migrate in solvent 1 but did comigratewith disialyllacotse isolated from bovine colostrum in solvent 2 usedfor separation of oligosaccharides (not shown). The decreasedhydrophobicity of cleaved GD3 compared to native GD3 allowed itsseparation by HPLC on C18 reversed phase columns. Utilizing isocraticelution with methanol, cleaved GD3 with proteins resulted in formationof Schiff-bases between the modified ganglioside and e-aminolysilgroups. They were reduced to form stable secondary amine bonds betweenthe ganglioside and the protein by using sodium cyanoborohydride (Borchet al., 1971). The reducing agent was selective, and aldehyde groupswere not reduced in phosphate buffers at pH=6.5-7.5. The reaction wasmonitored by HPTLC and changing ration between cleaved GD3 and aresorcinol positive band that appeared at the origin was seen. This bandindicated the formation of the neo-glycoconjugates. The reaction wasnormally completed after incubation for 48 h. at 37° C. Disialyllactosewas readily removable by dialysis, excess cleaved GD3 by passage througha detergent removing column. The degree of coupling was determined bysialic acid and protein determinations. The weight ratio of GD3 toproteins in the conjugates depended on the accessibility of lysinegroups in the different proteins and is given in Table 1. The averageyield of GD3 coupled to proteins overall was 30%.

The carbohydrate part of GD3, disialyllactose, was coupled to proteinsutilizing two different methods. The conjugation of disialyllactose, wasperformed by reductive amination resulting in the open ring form of theglucose conjugated to proteins (Gray et al., 1978). The method requireda long incubation period of the oligosaccharides with proteins andyields were less than 20%. The second oligosaccharide conjugation method(Roy and Laferriere 1990) resulted in a closed terminal glucose ringcoupled to proteins. TABLE 1 Reciprocal ELISA titer against GD3 GD3/ No.of Protein Vaccine mice weight ratio IgG IgM GD3 5 — 0(5) 20(3), 0 (2)GD3-ganglioside conjugate: GD3-KLH^(a) 14 0.69 10240(2), 2560, 5120(2),1280(2), 2560(3), 640, 1280(2), 320(3), 80, 40 160(2), (2), 0 80(3), 20,0 GD3-cBSA 15 0.77 2560(2), 80(2), 320(2), 40(2), 160, 80 20(7), (2), 400(4) (4), 20 (2), 0(2) GD3-OMP 15 0.93 2560, 80 1280, (4), 20 320(2),(3), 0(7) 160(7), 80(4), 40 GD3-MAP 10 1 40, 0(9) 160(2), 40(4), 20(3),0 GD3-Polylysine 10 n.d.^(b) 0(10) 320, 160 (4), 80, 40, 20 (2), 0GD3-oligosaccharide conjugate: Disialo- 4 0.055 0(4) 160(3), KLH^(c) 80Disialo- 4 0.16 20, 0(3) 40, 20 cBSA^(c) (3) GD3-KLH^(d) 4 0.25 20, 0(3)40(2), 0(2) GD3-cBSA^(d) 4 0.34 0(4) 0(4) GD3-Polylysine^(d) 5 n.d.^(b)0(5) 80(3), 40(2)^(a)KLH: keyhole limpet hemocyanin, cBSA: cationized bovine serumalbumin, OMP: meningococcal outer membrane proteins, MAP: multipleantigenic peptide, Polylysine: poly L-lysine^(b)n.d.: not done^(c)open ring^(d)closed ringSerological Response Against GD3 after Vaccination with GD3-ProteinConjugate Vaccines

Preimmunization sera did not show IgM or IgG reactivity with GD3.Immunization with 20 ug GD3 alone or mixed with 10 ug of the adjuvantQS-21 failed to induce GD3 antibodies (Table 1). Some groups of miceimmunized with 20 ug of GD3 conjugated to proteins plus 10 ug QS-21showed increased immune responses against GD3. GD3-poly-L-lysineconjugate, representing a high density of GD3 epitopes, induced amoderate titer IgM response (range 1/20-1/320) and no IgG response.GD3-conjugated to outer membrane proteins of Neisseria meningitidis(GD3-OMP), also induced moderate titer IgM (rang 1/20-1/320) and lowtiter IgG (range 1/20-1/80). Only one mouse showed high titer IgMresponse of 1/1280 and high titer IgG of 1/2560 after vaccination withGD3-OMP. GD3 conjugated to cationized BSA(GD3-cBSA), showed low titerIgM by ELISA (range 1/20-1/80) and high titer IgG(range 1/20-1/2560).The synthetic MAP peptide, containing a malarial T-cell epitope,provided 8 free aminogroups at its aminoterminal end and 4 were able tobe conjugated to GD3. GD3-MAP induced low titer IgM (range 1/20-1/160)and only one mouse produced low titer IgG response of 1/40. GD3conjugate to KLH (GD3-KLH) induced the highest response compared toother conjugates, with highest titer IgM (range 1/20-1/2560), as well ashighest titer IgG (range 1/40-1/10240) response. Immunization with bothtypes of disialyllactose protein conjugates induced only low titer IgMthat was cross reactive with GD3 ganglioside (range 1/20-1/160) and nosignificant IgG response.

Specificity of GD3 Reactive Sera by Immune Thin Layer Chromatography

Immune thin layer chromatography (ITLC) allows testing of GD3 antiseraon human tissue ganglioside extracts and to determine specificity totumor derived gangliosides. Examples of ITLCs with human tissue extractsand high titer IgM and IgG sera induced by immunization with GD3-KLHconjugate are shown in FIGS. 3 a and 3 b. Sera were tested at a 1/150dilution against ganglioside extract of human brain, neuroblastoma,melanoma, as well as against the immunogen GD3 (bbm) isolated frombovine buttermilk. The reactivity on ITLC was compared with resorcinolstained HPTLC which shows the total ganglioside composition found inthese tissues. Normal brain predominantly contains GM1, GD1a, GD1b andGT1b, while the neuroblastoma extract contains in addition the majorgangliosides GD2 and GM2 and the melanoma extract contains mainly GM3and GD3. IgG antisera showed specific reactivity only with GD3 in allthree tissues extracts tested (FIG. 3 a), as did the control mAb R24.IgM antisera (FIG. 3 b) on the other hand showed some cross reactivitywith structurally related gangliosides and sulfatide in brain extract.Immune responses induced by vaccination with other GD3 conjugates showedthe same specific reactivity, but were weaker and more concentratedantisera had to be used (not shown). High titer antisera identified byELISA in mice immunized with GD3-cBSA showed high background by ITLC. Avariety of blocking agents were used with these sera unsuccessfully. Nospecific reactivity with GD3 in tissue extract could be detected.

Specificity of GD3 Reactive Sera by Dot Blot Immune Stains

The specificity of all high titer IgM and IgG antisera (by ELISA >1/160)was studied with purified gangliosides GM3, GD2, GD1b, GD3 and GT3isolated from bovine brain or buttermilk, and with GD3 isolated fromhuman melanoma tissue. These structurally related gangliosides werespotted onto nitrocellulose strips in similar amounts and reacted withimmune sera. A sample of dot blot immune stain experiments with seraobtained before and after immunization of mice with GD3-KLH and GD3-OMPis shown in FIG. 4. Presera did not show any reactivity with thesegangliosides. Sera obtained after immunization with GD3-KLH showedspecific IgM and IgG reactivity with GD3 from bovine buttermilk (theimmunogen) as well as with GD3 isolated from human melanoma tissue. Insome cases cross reactivity with GT3 was seen, a reaction observed alsowith the positive control mAb R24 (Houghton et al., 1985). High titersera from mice immunized with GD3-cBSA showed only background reactivitybut no specific reactivity against any ganglioside was detected (notshown). Dot blot reactivity induced by other GD3 conjugates werespecific for GD3 (not shown). The results indicate that specific hightiter IgM and IgG responses can be induced in mice with GD3-proteinconjugates, and that the strongest reactivity was induced with GD3-KLHconjugates. The conjugated method seems to preserve the importantepitopes on the GD3-oligosaccharide chain and GD3-conjugates did notinduce cross reactivity with structurally related gangliosides.

Cell Surface Reactivity of Immune Sera Determined by FACS Analysis:

Sera from mice were tested for binding to cells of the melanoma cellline SK-MEL-28, a cell line known to express cell surface G_(D3). Arepresentative example of a FACS analysis utilizing a fluoresceinisothiocyanate-labeled secondary goat anti-mouse antibody is shown inFIG. 5. Sera before and after immunization with G_(D3)-KLH and QS-21were tested. Preimmunization serum stained 8% of the target cells,postimmunization serum 92%.

Discussion

An approach for construction of ganglioside conjugate vaccines isdescribed here to 1) establish a coupling reaction with proteinsapplicable to different tumor gangliosides, 2) increase theimmunogenicity of GD3 as the major ganglioside associated with melanomaand, 3) define the most effective protein carrier. Gangliosideconjugation must be accomplished without altering the immune dominantcarbohydrate moiety. It has been shown that modification of GD3 in itscarbohydrate portion for example conversion of carboxyl groups to amidegroups, increases the immunogenicity of the synthetic antigens but therewas no significant cross reactive antibody response with native GD3(Ritter et al., 1990b). Consequently, this approach aimed at couplingGD3 via its ceramide portion without alteration of the carbohydratepart. The ceramide, characteristic for all gangliosides, was cleavedwith ozone at the C4 position of the sphingosine base and a functionalaldehyde group was introduced. Coupling to proteins was realized byreductive amination to form a stable amine bond between ganglioside andε-aminolysyl groups of proteins. Cleavage of gangliosides by ozonolysisand subsequent conjugation has not yet been described and it was assumedthat the aldehyde intermediate of gangliosides is instable.Fragmentation has been reported, when initiated by the attack of hydroxyions under alkaline conditions, migration of double bond occurs andβ-elimination causes release of the oligosaccharide part (Kanfer andHakomori, 1983; Wiegandt and Baschang, 1965). The aldehyde function isfound to be sufficiently stable at neutral pH, Schiff bases with aminogroups of proteins are readily formed and β-elimination occurs only to asmall extend. An overall yield of 30% was comparably efficient asdescribed for the conversion of gangliosides into lyso-derivatives(Neuenhofer et al., 1985). The aldehyde derivative of GD3 did not reactany longer on immune thin layer chromatography (ITLC) with mAb R24. Asimilar phenomena has been described in connection with the reactivityof mAb M2590 with GM3 and reactivity was dependent on the acyl chainlength (Itonori et al., 1989). On the other hand, GD3 proteinconjugates, showed reactivity with mAb R24 by ITLC and western blot,indicating that immune dominant epitopes were restored in the GD3neoglycoconjugates.

Once the conjugation method for generation of ganglioside vaccineestablished, appropriate carrier proteins had to be selected. Lowell etal. (1988) described an elegant vaccine system that induced high titerantibody responses by complexing of bacterial carbohydrate and peptideantigens via a synthetic, hydrophobic foot into outer membrane proteins(OMP) of Neisseria meningitidis and effective without additionaladjuvant (Donnelly 1991). This system was directly applicable togangliosides due to their amphipatic nature. In previous experiments,applicants absorbed gangliosides by hydrophobic interaction onto theseproteins and were able to induce high titer IgM responses (Livingston etal., 1993b). Covalent attachment was utilized, but GD3-OMP conjugateinduced only occasional IgG responses and the IgM response did notexceed results of previous trials without conjugation of GD3. CationizedBSA, which has been reported to be a potent immune modulator for proteinantigens (Apple et al., 1988), was able to enhance specific immuneresponse to poorly immunogenic proteins after conjugation. GD3-cBSAconjugates induced only moderated IgM response, but high titer IgGantibodies were analyzed by ELISA. Further examination of these hightiter antisera by ITLC or dot blot immune stains indicated that theresponse was not specific for GD3. Another appealing approach forvaccine construction has been described by J. Tam et al. (Tam, 1988; Tamand Lu, 1989) as a multiple antigenic peptide system (MAP). Based on anoligomeric branching lysine core, MAPs consist of four or eightdendritic peptide arms containing B- and T-cell epitopes. The immuneresponse to peptides was dramatically increase when these constructswere used in comparison to the peptides with B-cell or T-cell epitopesalone. When GD3 was attached to the amino terminal end of a MAPstructure, containing a malarial T-cell epitope, only moderate IgM andno IgG response against GD3 was detected. Although this approach is veryeffective for synthetic peptides it seems to be of litter use forgangliosides vaccines. It has been reported, that anti gangliosidesantibodies can distinguish between tumor derived GM3 and GM3 on normaltissue because of their different cell surface density (Nores et al.1987). The conjugation of GD3 to polylysine was thought to represents ahigh density of GD3 epitopes combined on a single molecule. The responseto GD3-polylysine was moderate, only medium titer IgM response wasdetectable and no IgG response. Finally, the mice immunized with GD3conjugated with keyhole limpet hemocyanin, GD3-KLH, were able togenerate the highest titer IgM and IgG responses and significantlyhigher than those generated by previous vaccines.

These sera when tested by immune stains assays were found to be highlyspecific for GD3 in human tissue extracts. Time course experiments ofthe IgM immune response indicated similar characteristic as observed inprevious trials (FIG. 2). IgM peak titer were received after the thirdvaccination when administered in biweekly intervals. The responsedeclined fast and continuous vaccination did not induce a significantboost in antibody response. This is the first report to show inductionof high titer IgG response using ganglioside vaccines. This responselasted significantly longer than IgM response and was boosted bycontinuous vaccination, but was not comparable to the exponentialpotentiation of response often seen with protein antigens. The subclasswas determined as mainly IgG1 and it is not clear if T-cell dependentpathways were activated with ganglioside conjugate vaccines. Althoughthe importance of T-cell help in B-cell maturation is undoubted, theregulation of antibody class is controversial and several reports haveshown that isotype switch is possible with T helper cell activity (Tealeand Abraham, 1987). Conjugates containing solely the oligosaccharidepart of GD3 were found not to be reactive with mAb R24 and were not ableto induce a significant immune response against GD3 ganglioside.Modification of the glucose at the reducing end of the oligosaccharidechain during conjugation or the missing part of the ceramide mayinfluence the proper epitope presentation and the detection by theimmune system. Both methods used for conjugation were less efficient andyields were low. The induction of a specific immune response againsttumor associated gangliosides with less effective vaccines in patientsinduced already immune responses and were associated with betterprognosis. Ganglioside conjugate vaccines showed their ability to inducelong lasting and specific IgG response in mice with suggest, thatespecially GD3-KLH conjugate may soon prove usefulness as tumor vaccinein melanoma patients.

Second Series of Experiments

A Phase I trial of the immunological adjuvant QS-21 in melanoma patientsvaccinated with the ganglioside GM2 covalently attached to KLH.

Objective: To determine the optimal safe dose of the immunologicaladjuvant QS-21 for induction of antibodies against GM2

Background

Patients with AJCC Stage III melanoma have a recurrence rate at twoyears and mortality rate at three years of 60-70% (Hilal et al. 1981;Eilber et al. 1976). Patients with Stage IV melanoma who are free ofdisease after surgery have a more ominous prognosis. There is notreatment known to alter these rates. The standard treatment for StageIII melanoma after surgery is close observation.

Some patients with melanoma have antibodies in their serum which reactwith highly restricted melanocyte differentiation antigens have beenshown. In some case, it was noted that the presence of these antibodieshas been associated with an unexpectedly favorable course (Livingston etal., 1987). As only few patients have these antibodies in their serum,attempts have been made to induce antibody formation by immunizing thepatients with melanoma vaccines containing the relevant antigens.Vaccine prepared form whole cells have been ineffective in this regard(Livingston et al. 1982). Purified antigens, rather than whole melanomacells are now proposed for vaccine production. In recently completedtrials, patients have been vaccinated with BCG-GM2 and short-lived IgMantibody production were seen in 33 of 44 patients (Livingston et al.1989; Livingston, 1989), but IgG antibody responses were rarely seen.

Potent adjuvants or other approaches for increasing the immunogenicityof gangliosides such as GM2, and in particular for inducing an IgGresponse are continuously sought. It was found to be most successful atinducing an IgG response to gangliosides in the mouse by covalentattachment to keyhole limpet hemocyanin. The basis for this is theconcept of split tolerance. Studies of immunological tolerance and ofways to overcome it have shown that in a variety of experimental systemsT cell unresponsiveness is more rapidly induced and more easilymaintained than B cell unresponsiveness (Romball et al. 1984; Weight,1977). Levels of circulating antigen suitable for maintaining T celltolerance frequently fail to maintain B cell tolerance. Consequently, ifT cell help is provided (as by potent irrelevant antigens such as KLHcovalently attached to the desired immunogen), antibodies can be inducedto tolerated T cell dependent antigens. This approach has beensuccessfully used to induce IgG antibodies against a variety ofcarbohydrate antigens in experimental animals (Kundu et al. 1980; Gray1978; Chang and Rittenberg, 1981; Longenecker et al., 1987) and recentlyagainst H. Influenza Polysaccharide antigen in infants.

The molecular weight of KLH is quite variable but approximate 2×10⁶daltons. It has been injected intradermally in patients, by severalinvestigators (Berd et al., 1982) at a dose of 1 mg to induce delayedtype hypersensitivity (DTH). Pyrogen free KLH has been prepared byBiomira Inc. (Edmonton, Canada) and covalently linked to GM2 at a highepitope density (1000/l). High titer IgG responses against GM2 usingthese preparations mixed with immunological adjuvants in the mouse havebeen induced.

Of the immunological adjuvants tested in preclinical studies withKLH-conjugate vaccines such as T antigen-KLH, QS-21 has been the mosteffective. IgG antibody titers over 1/4000 and potent DTH are seen inmost mice. T-KLH alone results in a median titer of 1/160 with no DTH.QS-21 is a carbohydrate extracted from the bark of the South Americantree Quillaja saponaria Molina. The monosaccharide composition,molecular weight, adjuvant effect and toxicity for a series of thesesaponins has been described (Kensil et al. 1991). QS-21 was selected dueto its adjuvanticity and lack of toxicity. It has proven nontoxic andhighly effective at augmenting the immunogenicity of an FeLV subunitvaccine in cats (Marciani et al.) and an HIV-1 recombinant vaccine inRhesus monkeys.

In addition, as it was shown that some patients with melanoma havesuppressor cells which may interfere with immunization and that thesecells can be inhibited by a low dose of cyclophosphamide (Livingston etal., 1987b), each patient will receive a low dose of cyclophosphamideprior to the first vaccination. This combined approach has been found toaugment the immunogenicity of glycolipids and other antigens inexperimental animals and melanoma patients (Livingston et al. 1987a;Livingston et al., 1989).

Study Population

Patients with high risk AJCC stage III or IV malignant melanoma two toeight months after surgical resection, whose pathology slides have beenreviewed by the Memorial Hospital Department of Pathology, and who areclinically free of disease will be eligible. They must have aperformance status of >80 (Karnofsky) and an expected survival (asidefrom their melanoma) of at least 5 years. Pregnant women, patients withallergies to seafood and patients with creatine or bilirubin >2.0 areexcluded. Patients may have received previous irradiation, chemotherapyor immunotherapy (completed 8 weeks prior to vaccination).

Treatment Evaluation

Patients must have had a thorough physical examination at MemorialHospital and chest X-ray, CBC, serum creatinine and liver function testswithin 3 weeks of treatment. patients with abnormal LFT or chest X-rayresults are accepted if further tests (i.e. CTT, tomograms, etc.) showno melanoma.

Vaccine Preparation

Chemistry and Manufacturing

Drug Substance

Name and Source

Proper Name:

GM2-KLH synthetic tumor associated glycoconjugate (S-TAG)

-   -   to be used for active specific immunotherapy        GM2-HSA synthetic tumor associated glycoconjugate (S-TAG)    -   to be used for skin testing of patients undergoing active        specific immunotherapy with the GM2-KLH.        Chemical Name:        11³NeuAc-GgOse₃Cer-keyhole limpet hemocyanin (KLH)        Laboratory Codes:        GM2-KLH Lot # 5        GM2-HSA Lot # 1        Manufacturer: Biomira Inc. Research Centre One, Edmonton        Research and Development Park, 9411-20 Avenue Edmonton, Alberta        T6N 1E5 Canada.

Materials Used for the Preparation of the GM2 Hapten MATERIAL SUPPLIERGRADE Acetone BDH ACS Ammonia Solution BDH ACS Chloroform BDH ACSEthanol Commercial — Alcohol Ltd. Ethyl Ether BDH ACS Methanol BDH ACS2-Propanol Fisher UN1219 ACS Water Travanol sterile water forirrigation. Calcium Chloride Fisher Certified (anhydrous - 20 meshgranular) Dimethyl Sulfide Aldrich 99%+ GM2 Fidia — Oxygen Linde UN1072UHP Silica Gel E Merck Kieselgel 60H Art 7736 Sodium Aldrich 95% PureCyanoborohydride TLC Plates E Merck Kieselgel 60H F254

Materials Used in the Conjugation Procedure MATERIAL SUPPLIER GRADEKeyhole limpet hemocyanin Calbiochem, --- (KLH), lyophilized, 60% SanDiego, CA protein in BES [N,N-bis- (2-hydroxyethyl)-2-aminoethanosufonic acid] buffer, purity 90% Deoxycholic acid, sodium salt AldrichAnalytical (DOC) (monohydrate) 98% Ethylenediamine tetraacetic AldrichACS acid di-sodium hydrogen orthophosphate BDH Analytical (anhydrous)(Na₂HPO₄) Sodium chloride BDH Analytical (NaCl) Potassium dihydrogen BDHAnalytical orthophosphate (KH₂PO₄) Sodium hydroxide BDH Analytical(NaOH) Tris (hydroxymethyl) Sigman — aminomethane hydrochloride Sodiumcyanoborohydride Aldrich — (NaBH₃CN) Sepharose CL-4B Pharmacia —Nitrogen gas (filtered) Medigas — Human serum albumin, 25% Miles USP,For solution (HSA) injection GM2 aldehyde Biomira Inc. —

Development Chemistry Data for the GM2 and GM2 Aldehyde:

The structures of GM2 and GM2 aldehyde were characterized by BiomiraInc. by ¹H NMR spectroscopy, thin layer chromatography (TLC), FAB-MS andFT-IR. STRUCTURAL MOLECULAR MOLECULAR FORMULA FORMULA WEIGHT GM2 -ganglioside (compound #1) GalNAcB1-4GaLB1-4 C₆₇H₁₂₁O₂₆N₃ M − 1 = 1382Solid GlcB1-1Cer Neu5Aca2 I³NeuAc-GgOse₃Cer C₆₉H₁₂₅O₂₆N₃ M − 1 = 1410(acid) TLC: Rf = 0.21(65:35:8 CHCL₃—CH₃OH—H₂O) Rf = 0.60(5:4:1CHCl₃—CH₃OH-0.2% aqueous CaCl₂) Rf = 0.2(7:1:1 (CH₃)₂CHOH—NH₄OH—H₂O)STRUCTURAL MOLECULAR MOL. PHYSICO-CHEMICAL FORMULA FORMULA WT.CHARACTERISTICS GM2-aldehyde C₅₃H₉₃O₂₇N₃ 1204.29 Cream White, (compound#2) Odorless, Amphorous SolidSTRUCTURAL DATA¹H(DMSO-d₆:D₂)δ:9.48(d, 1H, J=2, OHz), 4.79(d, 1H, J=8, OHZ, I-1),2.54(dd, 1H, A-3e), 1.88(s, 3H, Ac), 1.78(s, 3H, Ac), 0.85(t, 3H,J=6.6Hz, CH₃).FT-IR (KBr Cast, CM⁻¹): 3439, 3420, 2952, 2923, 2851, 1634, 1070(possibly the gem diol).TLC Rf=0.5 (5:4:1 CHCl₃—CH₃OH-0.2% aqueous CaCl₂)Data for the KLH, GM2-KLH, HSA and GM2-HSA:

The keyhole limpet hemocyanin (KLH) is a large, complex protein composedof a number of smaller molecular weight subunits. The KLH is extractedand purified from the keyhole limpet mollusk (Megathura crenulata). TheKLH, HSA and the conjugates were characterized by Biomira Inc. bySepharose CL-4B gel filtration chromatography, isoelectric focusing(IEF) and the color metric resorcinol-hydrochloric acid method (1).RESORCINOL-GEL SEPHAROSE CL-4B ISOELECTRIC HCl (# moles CHROMATOGRAPHYFOCUSING moles of hapten/ COMPOUND Molecular Weight (daltons) (Isoelec.pts.) moles of protein) KLH Whole mol.(2): >2 × 10⁶ Mult. bandsSubunits: 2-7 × 10⁵ between pH 4.65 — and pH 6 GM2-KLH Whole mol.(2) >2× 10⁶ Multiple bands Subunits: 2-7 × 10⁵ between pH 4.65  200-1400 andpH 6 HSA 5-9 × 10⁴ Broad band — at pH 4.65 GM2-HSA 5-9 × 104 Broad band 2-12 at pH 4.651. L. Svennerholm, Biochimca et Biophysica Acta, 24, (1957), 604-611.

-   2. Using the Sepharose CL-4B gel filtration method, the whole KLH    protein molecule elutes in the void.    -   volume of the column which indicates that the molecular weight        of KLH is >2×10⁶. This value is consistent with the range of        weights given in the literature for this protein.        GM2-KLH Manufacturing Flow Chart

Stage 1—Purification of GM2: GM2 (FIDIA) sent for viral testing ↓ SilicaGel Column Chromatography 1. 65:35 chloroform - methanol 2. 65:35:4chloroform - methanol - water ↓ In-process QC: 1. TLC 2. ¹HNMR STAGE 2 -PURIFICATION OF THE KLH: KLH dissolved in PBS pH 7.5 (˜3 mg/ml) ↓Centrifuged ↓ Sample of dissolved KLH run through Sepharose column todetermine the molecular weight profile ↓ Diafiltered v.s the followingbuffers successively: 1. PBS pH 7.5 2. TRIS-HCl, EDTA pH 7.75 3.TRIS-HCl, EDTA, 0.5% DOC pH 7.75 4. TRIS-HCl, EDTA pH 7.75 5. PBS pH 7.5↓ Volume adjusted with sterile, pyrogen-free PBS to ˜75 mL ↓ Centrifuged↓ Sterile Filtered ↓ BioRad Protein Assay Performed ↓ Sample of the KLHrun through a Sepharose column to determine molecular weight profile ↓Concentration adjusted to 10 mg/mL with PBS pH 7.5 ↓ KLH aliquotted inserum vials and frozen at −20 ± 5° C. ↓ In-process tests: 1. Isoelectricfocusing (IEF) 2. Limulus amebocyte lysate (LAL) pyrogen test ↓ STAGE3 - SYNTHESIS OF GM2 ALDEHYDE (COMPOUND #2): GM2 (Compound #1) ↓ (1) O₃,MeoH ↓ (2) CH₃SCH₃ ↓ GM2 Aldehyde (Compound #2, may be the gem diol) ↓In-process tests done in-house: 1. TLC ↓ STAGE 4 - CONJUGATION OF THEGM2 HAPTEN TO KLH: Sterile pyrogen free KLH thawed ↓ KLH added to haptenin 4:1 ratio (w/w) ↓ Incubated at room temperature with shaking for 3minutes ↓ NaBH₃CN is added to hapten/KLH mixture in 1:1 ratio (w/w) tohapten ↓ Reaction mixture is gently stirred at room temperatureovernight then at 40° C. for 4 days ↓ STAGE 5 - DIAFILTRATION OF THECONJUGATE: Conjugate is diafiltered vs.: -PBS pH 7.5 -TRIS/EDTA pH 7.75-TRIS/EDTA/0.05% DOC pH 7.75 -TRIS/EDTA pH 7.5 -PBS pH 7.5 Conjugationaseptically removed from the Amicon filtration unit ↓ Centrifuged ↓Conjugate sterile filtered ↓ In-process QC tests: 1. BioRad ProteinAssay 2. Sepharose gel filtration 3. Isoelectric focusing (IEF) ↓Concentration of conjugate aseptically adjusted to 1 mg/mL ↓ Conjugatedispensed into 1 mL sterile, pyrogen free serum and frozen at −20 ± 5°C. ↓ Final QC testing: 1. Enzyme immunoassay (EIA) 2. LAL pyrogen test3. BioRad protein assay 4. Resorcinol-HCl assay 5. Rabbit pyrogen test6. General safety test 7. Sterility test 8. Impurity test for cyanideMethod of Manufacture of GM2-KLH ConjugatesThe manufacturing of the GM2-KLH semisynthetic glycoconjugate fir ASIand GM2-HSA semi-synthetic glycoconjugate for skin testing is carriedout in 5 stages:

-   1. Purification of incoming GM2 (bovine source) (compound #1).-   2. Purification of keyhole limpet hemocyanin (KLH).-   3. Synthesis of GM2 aldehyde (compound #2).-   4. Conjugation of the GM2 hapten to KLH.-   5. Diafiltration of the conjugate.    Stage 1: Purification to GM2 (Compound #1):    -   Name: GM2 ganglioside    -   Abbreviated Name: II³NeuAc-GgOse₃Cer

A sample of GM2 ganglioside (bovine source) starting material suppliedby FIDIA is sent for viral testing (8CFR protocol). All glass ware iswashed with distilled acetone followed by distilled ethanol and thenoverdrive (130° C.) for 18 hours prior to use. A column (Michel-Miller S795-10) of silica gel (30.5 g, Kieselgel 60H, Art 7736, E. Merck) ispacked at 75 psi (SSI Model 300 Lo pump) using 65:35 chloroform:methanolas solvent. GM2 (200 mg) is applied as a concentrated 65:35chloroform-methanol solution and elution is performed with this solvent,followed by 65:35:4 chloroform-methanol-water. The fractions areanalyzed by TLC (Rf 0.6, 5:4:1 chloroform-methanol-0.2% aqueous CaCl₂).The GM2 containing fractions are pooled and evaporated to give a creamywhite amorphous solid.

In-process testing for this material (compound 1 includes ¹H NMR andthin layer chromatography (TLC) to confirm the identity and purity ofthis ganglioside. The in-process test results must meet thespecifications listed under developmental chemistry. If this material isfound to be impure, the above purification is repeated.

Stage 2: Preparation of Sterile, Pyrogen-Free Keyhole Limpet Hemocyanin(KLH)

Preparation of KLH:

This entire procedure is carried out inside of a Class 100 biologicalsafety cabinet. Key hole limpet hemocyanin (KLH) supplied by Calbiochem,is dissolved in 100 mL of sterile, pyrogen-free phosphate bufferedsaline (PBSP pH 7.5. This solution is incubated at 2-6° C. for 18 hoursto allow the KLH to dissolve into solution. The solution is then spun at200 rpm for 30 minutes. The supernatant is collected and a sample ofthis is run through a Sepharose CL-4B gel column to determine themolecular wight profile of the unprocessed KLH.

Prior to dialysis of the KLH, the Amicon Stirred Ultrafiltration Cell ismade sterile and pyrogen-free by rinsing it four times first withsterile water for injection (WFI) then filling it with 95% ethanol andletting it stir for 2 hours. The unit is again rinsed with WFI waterthen autoclaved.

The supernatant containing the KLH is poured into the sterile,pyrogen-free 400 mL Amicon diafiltration unit with a YM 30 (30,000molecular weight cutoff) filter. The total volume of the KLH is thenbrought up to 350 mL with sterile, pyrogen-free or low pyrogen contentbuffers successively:

-   1. 1 complete change of PBS pH 7.5 (sterile, pyrogen-free)-   2. 3 complete changes of TRIS-HCl, EDTA pH 7.65 (sterile, low    pyrogen content)-   3. 2 complete changes of TRIS-HCl, EDTA pH 7.75 with 0.5%    Deoxycholic acid (DOC) (sterile, low pyrogen content)-   4. 4 complete changes of TRIS-HCl, EDTA pH 7.75 (sterile, low    pyrogen content)-   5. 3 complete changes of PBS pH 7.5 (sterile, pyrogen-free)

Each buffer change consists of bringing the volume in the Amicon unitdown to 50 mL or less then adding buffer to raise the volume back up to350 mL.

(The sterile, pyrogen-free PBS is prepared using chemicals that arebaked at 180-185° C. for 4.5 hours. The chemicals are added to sterilewater for injection (WFI) and mixed in a sterile, pyrogen-freecontainer. The chemicals or the other buffers cannot be baked todepyrogenate them as they melt as such extreme temperatures, therefore,these buffers are prepared in sterile WFI water in sterile, pyrogen-freecontainers and sterile filtered with a 0.22 μm filter. The pH of the PBSand TRIS-HCl buffers is adjusted to the required pH using sterile,pyrogen-free 2N sodium hydroxide.)

The DOC in the TRIS, EDTA pH 7.75 buffer serves to break the pyrogensdown into their lower molecular eight subunits which pass through thefilter while the KLH protein is retained in the Amicon unit (8.9).

The KLH solution is aseptically removed from the Amicon unit and spunagain at 2000 rpm for 30 minutes. The solution is then transferred to asterile, pyrogen-free graduated cylinder and the final volume isadjusted to 75 mL with sterile, pyrogen-free PBS pH 7.5.

The supernatant is then sterile filtered with a 0.22 μm low proteinbinding filter. A sample of the KLH is run through a Sepharose CL-4Bcolumn to determine if the treatment of the KLH with the buffers (theDOC in particular) affected the molecular weight profile of the KLHcompared with the initial column chromatography results of the untreatedKLH. The profile should not have changed significantly. An aliquot istaken of the KLH and a BioRad protein assay performed using KLH for thestandard curve. The final volume of the KLH solution is asepticallyadjusted with sterile, pyrogen-free PBS 7.5 to provide a final proteinconcentration of 10 mg/mL.

An LAL test is done to determine the level of pyrogens present in thepurified KLH. The pyrogen content must be less than 10 EU/mg for the KLHto be used in the conjugation procedure.

Isoelectric focusing (IEF) is done to check the purity and identity ofthe KLH. Past lots of KLH are run in parallel to act as standards.

The KLH solution is dispensed in 10 mL aliquots into sterile,pyrogen-free 30 mL serum vials and capped with sterile, pyrogen-freebutyl stoppers. The KLH is then frozen at −20±5° C. until time forconjugation to the hapten.

Stage 3: Synthesis of GM2 Aldehyde (of Gem Diol, Compound #2):

All glassware is rinsed with distilled methanol and overdried (130° C.)for 18 hours prior to use. A solution of the purified GM2 ganglioside(compound #1) (40 mg) in distilled methanol (10 mL) is stirred at −15°C. (dry ice-ethanol) and ozone gas (Orec O3V10-0 ozonator) is passedthrough the solution for 7 minutes. A stream of argon is then passedthrough the solution while the reaction is checked by TLC (5:4:1chloroform-methanol-0.2% aqueous CaCl₂). The solvents are then removedunder reduced pressure and the resulting material is dissolved indistilled methanol. To this solution is added methylsulfide (200 ml) andthe reaction mixture is stirred at room temperature for one hour. Thesolvents are then removed and the residue is triturated with ethyl ether(4×25 mL). The resulting white solid (compound #2) is dried in vacuo for15 minutes to remove any remaining solvent and is then used directly inthe subsequent conjugation step.

Due to the unstable nature of the resulting aldehyde (B-elimination),compound #2 is identified on a routine basis only by TLC. The TLC of atypical run generally indicates the presence of a small amount ofsphinganine or phytosphingosine analog (same Rf as compound #1) and asmall amount of reducing sugar (Rf 0.32).

Stage 4: Conjugation of GM2 Hapten to KLH (or HSA):

All manipulations are done in a Class 100 biological safety cabinet.

Two vials, each containing 10 mL of the frozen sterile, pyrogen-free KLH(10 mg/mL), are thawed at room temperature immediately before use.

The KLH protein (16 mL) is aseptically measured and added to the flaskcontaining the lyophilized GM2 hapten and a magnetic stir bar. Thesolution is gently agitated at room temperature for 3 minutes until allof the hapten has gone into solution.

The sodium cyanoborohydride (NaBH₃CN) (40 mg) is added to the hapten/KLHsolution then the flask is sealed with a stopper equipped with a sterilefilter needle. The solution is gently shaken then incubated overnight atroom temperature. The solution is then further incubated at 40° C. for 4days.

Stage 5: Diafiltration of the Glycoconjugates (GM2-KLH):

The contents of the hapten/KLH reaction vial are aseptically transferredto the sterile, pyrogen-free Amicon ultrafiltration unit with a YM-30filter. Filtered nitrogen is used to provide an operating pressure of 16psi for the Amicon unit. The conjugate is then diafiltered against thefollowing sterile, pyrogen-free or low pyrogen content bufferssuccessively:

-   1. 2 complete changes of PBS pH 7.5 (sterile, pyrogen-free)-   2. 2 complete changes of TRIS-HCl, EDTA pH 7.75 (sterile, low    pyrogen content)-   3. 2 complete changes of TRIS-HCl pH 7.75 with 0.5% Deoxycholic acid    (DOC) (sterile, low pyrogen content)-   4. 4 complete changes of TRIS-HCl pH 7.75 (sterile, low pyrogen    content)-   5. 3 complete changes of PBS pH 7.5 (sterile, pyrogen-free)

The glycoconjugate is then aseptically removed from the filtration unitand spun at 2000 rpm for 30 minutes. The supernatant is _(o)then sterilefiltered with a 0.22 mm low protein binding filter.

A sample of the glycoconjugate is obtained and the following in-processQC tests are done:

-   1. Sepharose gel filtration-   2. Isoelectric focusing (IEF)-   3. BioRad protein assay

Based on the results of the protein assay, the final volume of theglycoconjugate is adjusted with sterile, pyrogen-free pH 7.5 to yield aprotein concentration of 1 mg/mL.

Inside of a Class 100 biological safety cabinet, the finalglycoconjugate is then dispensed in 0.5 mL aliquots with an overfillvolume of 0.1 mL into 1 mL sterile, pyrogen-free, clear, borosilicateserum vials with red rubber stoppers and frozen at −20° C. During thefilling procedure, the air inside the filing area is monitored byexposing two blood agar plates to the air near the work area inside ofthe hood for a minimum of thirty minutes. These plates are thentransferred to a 37° C. incubator and incubated for 1-2 days. The platesare then examined for any bacterial or fungal colonies.

The vials are placed inside of a box with a label indicating the productname, lot number and number of vials. The box is then sealed and a labelwith the same information is placed on the outside of the sealed box.The box is then placed in Quarantine in the fridge for 1-2 days until itcan be labeled. Once the final QC tests have been done, labels arerequested. The product is labeled by the manufacturing personnel thenthe labeling is verified by the Quality Control department or theRegulatory Affairs department. The final product file is then signed offby the manager of Regulatory Affairs and by the Vice President and COOof the Immunotherapeutics division. The product is then released andstored in a “Released Product” freezer at −20° C.

Each lot of GM2-KLH and GM2-HSA goes through the following Final QualityControl tests:

-   1. Enzyme Immunoassay (EIA)-   2. LAL pyrogen test-   3. BioRad protein assay-   4. Resorcinol-HCl carbohydrate assay-   5. Rabbit pyrogen test-   6. General safety test-   7. Sterility test-   8. Impurity testing for cyanide

GM2-KLH is prepared by Biomira Inc. (Edmonton, Alberta) and used underan IND with the U.S. Food and Drug Administration. The GM2/KLH molarratio is 800/1 (actual=200-1400) and the conjugate is supplied at aconcentration of 0.57 mg conjugate per 0.5 ml phosphate buffered saline(PBS). This represents approximately 70 ug of GM2 ganglioside and 500 ugKLH per 0.5 ml PBS. On the day of vaccination for the initial 5immunizations, 0.5 ml will be placed in an individual syringe andbrought to the clinic for administration. This represents a GM2 dose of70 ug, a dose found effective in previous studies with GM2 plus variousadjuvants. The final (sixth) immunization will contain one-half of thisdose, 35 ug GM2 and 250 KLH.

QS-21 is extracted by Cambridge Bioscience Inc. (Worcester, Mass.) fromQuillaja saponaria Molina tree bark by silica and reverse phasechromatography as previously described (16). The purified GM2-KLHconjugate and QS-21 are tested for sterility by standard culturetechniques in the bacteriology laboratory, for pyrogenicity in rabbitsand for safety in rabbits and mice. They are aliquoted and stored at −15to −25° C. On the day of vaccination, 570 ug GM2-KLH (or 285 ug for thesixth vaccination) is mixed with QS-21, placed in an individual syringe,labeled, and brought to the clinic.

Four doses of QS-21 will be used, 10, 50, 100 and 200 ug, each dilutedto a total volume of 0.25 ml in PBS. The first group of 3 patients willreceive 6 vaccines containing 10 ug QS-21, the next 3 patients 50 ugQS-21, the next 3 patients 100 ug QS-21 and the final 3 patients 200 ugQS-21. No patient will be entered at the next dose until all 3 patientsreceiving the previous dose have received at least two vaccinations. Ifno toxicity is seen at the 200 ug dose and if the immunologicalreactivity to GM2 antigen and KLH has not plateaued over the 50-200 ugrange, then 3 additional patients may be treated at a dose of 400 ug.Once a safe and maximally immunogenic dose has been identified, 6additional patients will be immunized at that dose to better define theantibody response.

The IND for the use of GM2-KLH plus QS-21 is held by MSKCC.

Treatment

Three to five days before the first immunization 200 mg/M² ofcyclophosphamide is administered IV. This is the dose and scheduleapplicants have used successfully in past vaccine trials. Fourvaccinations are then administered subcutaneously at two week intervals,beginning 2-30 weeks after surgical resection of all known disease. Twoadditional vaccinations are administered at two month intervals.

Evaluation

Serological response: The primary end point of this trial is serologicresponse. Peripheral blood (30 ml) will be drawn immediately before eachvaccination and 2 weeks and 5 weeks after the fourth, fifth and sixthvaccinations. Thereafter, blood may be drawn at 3-month intervals, aslong as detectable antibody against GM2 persists. Sera obtained 2 weeksafter the fourth, fifth and sixth vaccines from all patients will betested by ELISA for antibodies against GM2 and related gangliosides.Patients with titers of 1/80 or greater which are shown by ELISA andimmune thin layer chromatography on a variety of glycolipids to reactspecifically are considered serologic responders and additional serafrom serological responders may be tested to better define the antibodyresponse to vaccination. An additional 60 ml of peripheral blood may beobtained 2 weeks after the fourth vaccination for study of the immuneresponse against GM2 at the clonal level if applicants see evidence ofhigh titer IgG anti-GM2 antibody induction.

Supernatants from EBV-transformed lymphoblasts and subsequently producedhybridomas will be used for this purpose.

Delayed hypersensitivity response: Skin tests for delayedhypersensitivity against KLH, GM2 and GM2 attached to human serumalbumin, will be performed at the time of the fifth immunization.Reactions with more than 5 mm of induration at 48 hours will beconsidered positive and, if against GM2, will be studied further by skintests with a variety of gangliosides.

Clinical course: Patients will be evaluated at Memorial Hospital at thetime of their fourth and sixth vaccinations and a chest X-ray andscreening profile will be performed at the time of the sixthvaccination. Additional and subsequent follow-up will be performed bytheir oncologists.

Criteria for Cessation of Treatment

Regional recurrence routinely treated by local therapy (surgery orintralesional injection) is no reason for cessation of treatment if thepatient is rendered disease-free. Treatment will be discontinued if thepatient develops recurrent disease requiring systemic treatment orradiation therapy.

Statistical Considerations

This is a Phase I study primarily concerned with an evaluation oftoxicity and the IgG response against GM2 and KLH after vaccination.Three patients will be vaccinated at each QS-21 dose at 4 escalatingdoses. If the IgG response continues to increase with each escalation:the highest dose producing no more than grade I systemic or grade 2local toxicity will be selected, and if no toxicity is seen a fifth dosewill be tested. Based on studies in the mouse, it is expected that theIgG response to peak or plateau after the second or third dose. Once asafe and immunogenic dose range has been identified, 3-6 additionalpatients will be immunized at the dose that appears most immunogenic andat the next higher and lower doses to gain confidence in the degree oftoxicity and immunogenicity at these doses.

Risks

GM2-KLH: No toxicity was observed attributed to GM2 over 100 patientstreated with vaccines containing 200 ug GM2. KLH has been used at doesof 1 mg to immunize and test the immune status of over 50 patientswithout toxicity (15). 18 patients are immunized with vaccinescontaining GM2-KLH. One patient has experienced pain and tenderness atvaccines sites lasting 48 hours attributable to GM2-KLH, presumably adelayed type hypersensitivity response to KLH. No dose attenuation wasrequired. The only expected side effect of GM2-KLH is inflammation atvaccine sites lasting several days. If fever above 38° C. or severelocal toxicity that limits normal activity occurs, the dose of GM2-KLHin future vaccines may be decreased by a factor of 3.

Saponins have not been used in man. They are known to be hemolytic, butthe QS-21 fraction was selected because it has shown no clear toxicityat doses as high as 125 ug per mouse, cat and Rhesus monkey. On a M² orkg basis, this is thousands of times the maximal dose proposed here.

Autoimmune or hypersensitivity reactions to components of the vaccine,or to skin test antigens, are a theoretical, but remote possibility.

Criteria for Toxicity: Toxicity will be graded in accordance with theCommon Toxicity Criteria developed by the National Cancer Institute(NCI). These criteria are on file with the IRB.

Significance of Study

Augmentation of the immune response to cancer can be attempted by twobasic approaches-nonspecific immunopotentiation which constitutes thebulk of past and current efforts at cancer immunotherapy, and specificimmunization which has not really been evaluated in the treatment ofcancer but has contributed much to the control of infectious diseases.It is the knowledge of microbial antigen which has permitted thedevelopment of successful specific immunization against infections. Thelack of knowledge of human cancer antigens, on the other hand, hasprevented exploration of specific immunization in the context of canceras it should be explored, using vaccines of defined cancer-restrictedantigenicity and demonstrating their immunogenicity in cancer patients.Recent progress in the definition of melanoma cell surface antigens nowpermits investigation of specific immunization.

Results

GM2-KLH was used as antigen in Phase I trial with QS-21. AJCC Stage IIIand IV melanoma patients who were free of disease after surgicalresection of all known disease were immunized with 500 ug of GM2-KLH(GM2/KLH ratio 200-1400/1, GM2 dose 70 ug) plus 10, 50, 100 or 200 ug ofQS-21 in groups of 3 patients each. All patients have now received thefull 6 immunizations. No toxicity was associated with the 10 or 50 ugQS-21 doses. The 100 ug dose was also well tolerated, but each of the 3patients treated at this dose had at least 1 of the 6 vaccinations whichresulted in a palpable lump and tenderness for 24-48 hours and 2 ofthese patients had this occurrence after 2 of the 6 immunizations.

No systemic side effects were detected. The 3 patients treated at 200 ugper vaccine experienced tenderness and pain at the injection sites whichlasted 2-10 days and low grade fevers and flu-lie symptoms includingmild headaches and diffuse aches and pains lasting 8-24 hours after mostof the vaccinations. Serologic responses against GM2 and KLH for these12 patients are summarized at the bottom of Table 2. While the toxicitywith the 200 ug dose was not truly the maximum tolerated dose, it wassignificant toxicity and there was no evidence that it was associatedwith increased adjuvanticity compared to the 100 ug dose. An additional9 patients are currently immunized, 3 each at the 50, 100 or 200 ugdoses to confirm these toxicity and immunogenicity results. The IgM andIgG titers of the 6 patients receiving GM2-KLH plus 100 or 200 ug QS-21are shown in FIG. 6. It appears that GM2-KLH plus QS-21 (100 ug) is astrikingly immunogenic vaccine, far superior to GM2/BCG or GM2-KLH plusDETOX or BCG with regard to both the titers and the duration of IgM andIgG antibodies.

Having shown that the GM2-KLH and GD3-KLH plus QS-21 vaccines aresignificantly more immunogenic that the previous ganglioside/BCGvaccines, the immunogenicity of GD2, GD3, and GD3 lactone in these newconjugate vaccines will be tested in the clinic. The basis for selectingthese gangliosides as immunogens in melanoma is a quantitative analysisof twenty melanoma biopsies using immune thin layer chromatography toquantitate the levels of these gangliosides on melanomas and variousnormal tissues.

Six to twelve melanoma patients will be vaccinated with each of theseganglioside conjugates which have been prepared and covalently attachedto KLH. Conjugates which are shown to be immunogenic in these pilotstudies will then be pooled and tested in an additional group of 12patients as a prelude to a large multi-center trial aimed at determiningthe impact of a consistently immunogenic polyvalent melanoma gangliosidevaccine on recurrence rate and survival. TABLE 2 PEAK ANTIBODY TITERAGAINST GANGLIOSIDE GM2 AFTER IMMUNIZATION WITH GM2-KLH PLUS VARIOUSIMMUNOLOGICAL ADJUVANTS Number Antibody Titers Dot Blot Immune of byElisa stain Vaccine Patients IgM IgG IgM IgG GM2/BCG 6 640(2), 40, 0(5)3⁺(4), 1⁺(3), 0 160(3), 0 2⁺, 0 (3) GM2-KLH 6 160(2), 80 0(6) 3⁺(2),1⁺(2), 0 (2), 40, 0 2⁺(2), 0 (4) (2) GM2-KLH + 6 320, 160 160, 0 3⁺(2),2⁺, 0(5) Detox (4), 0 (5) 2⁺(3), 1⁺ GM2-KLH + 6 1280, 320 320, 20 3⁺, 2⁺1⁺(2), 0 BCG (2), 80 (2), 0 (3), 0 (4) (2), 0 (3) (2) GM2-KLH + 65120(2), 1280 3⁺(5), 3⁺(5), QS-21 1280(3), (2), 640, 2⁺ 2⁺ 320 320 160,80  10 ug 3 1280, 160, 80, 3⁺(2), 3⁺(2), 640, 40 0 2⁺ 2⁺  50 ug 3 640,320, 80(2), 3⁺, 2⁺ 3⁺, 2⁺(2) 160 40 (2) 100 ug 3 5120, 1280 1280, 3⁺(3)3⁺(3) (2) 640, 160 200 ug 3 5120, 1280, 3⁺(3) 3⁺(2), 1280, 320 320, 802+

Third Series of Experiments

The cell surface gangliosides GM2, GD2 and GD3 are often overexpressedin malignant melanoma. Applicants have shown previously thatimmunization of melanoma patients with GM2 and BCG induced an IgMantibody response in most patients, and that patients with high titerGM2 antibodies showed increased survival. As is commonly seen withcarbohydrate antigens (which are T-independent), the IgM response wasshort-lived, and an IgG response was rarely observed. To increaseimmunogenicity, applicants conjugated GM2 covalently with keyhole limpethemocyanin (KLH). GM2-KLH vaccine was given to melanoma patients withone of three adjuvants—BCG, DETOX or QS-21. The most effective vaccinewas GM2-KLH with QS-21. It induced a much higher titer and longerlasting IgM GM2 antibody response, and a consistent IgG response(isotype IgG1 and IgG3). It also induced the highest titer anti KLHresponse The results suggest that the conjugate GM2-KLH plus QS-21vaccine elicited significant T cell help. As there was no serioustoxicity, this vaccine approach is attractive for augmenting theimmunogenicity of other gangliosides such as GD2 and GD3 and fordetermine the effects of ganglioside antibodies on the course ofmelanoma. In addition, the finding that QS-21 significantly increasedthe immunogenicity of GM2-KLH suggests that it may do the same for otherconjugate vaccines, many of which are currently used without adjuvant.

Introduction

One of the changes that occur in the process of malignant transformationis an altered pattern of cell surface ganglioside expression in certaintypes of cancer, including malignant melanoma (1). In normalmelanocytes, GM33 is the predominant ganglioside. Other gangliosideswhich include GD3, GM2, GD1a and GT1b, constitute less than 10% of thetotal (2). In malignant melanoma, activation of glycosylating enzymesleads to increased expression of GD3, GD2, GM2 and 9-O-acetyl GD3 (3,4). These overexpressed gangliosides are attractive targets forimmunotherapy, including active immunization with ganglioside vaccines.In a series of studies involving GM2 vaccines in patients with malignantmelanoma, applicants have shown that vaccination (after low-dosecyclophosphamide and with BCG as adjuvant) induces IgM antibodies to GM2in most patients (5), and that disease-free interval and survival areextended in patients producing high-titer GM2 antibodies (6, 7).However, the induced antibody response to GM2 has the characteristics ofa T-independent response (predominantly IgM, short duration,inconsistent IgG response, lack of booster effect), and the othermelanoma gangliosides, GD3 and GD2, are not immunogenic whenadministered in the same way (8). As the relevant epitopes arecarbohydrates, applicants have explored approaches to increasingimmunogenicity that are suggested by the successful development ofcarbohydrate vaccines for bacterial infections. In the mouse, applicantshave shown that the immunogenicity of GD3 is markedly increased bycovalent binding to keyhole limpet hemocyanin, and that mice immunizedwith the GD3-KLH conjugate and the adjuvant QS-21 show a high-titer IgMresponse followed by a strong, long-lasting IgG response (9). Applicantshave now begun to test ganglioside conjugate vaccines in the clinic, andapplicants report here the initial results of immunizing patients withmalignant melanoma with a GM2-KLH conjugate vaccine.

Materials and Methods

Patients

Patients with malignant melanoma Stage III were considered eligible ifall metastatic disease had been rejected within the last 4 months. Noneof the patients had received prior chemotherapy or radiation therapy.

Vaccine Preparation and Administration

GM2-KLH vaccine: GM2-KLH conjugate was prepared by Biomira Inc.(Edmonton, Alberta) as described previously for GD3-KLH conjugatevaccine (9). Briefly, the conjugation procedure involved ozone cleavageof the ceramide double bond of GM2, introduction of an aldehyde group,and conjugation to aminolysyl groups of KLH by reductive amination. TheGM2/KLH molar ratio was approximately 800/1, and the vaccine wassupplied at a concentration of 0.57 mg conjugate in 0.5 ml normalsaline. This amount represented one patient dose and contained 70 μg GM2and 500 μg KLH. Groups of six patients each received GM2-KLH conjugatewithout adjuvant, GM2-KLH with DETOX, GM2-KLH with BCG and GM2-KLH withQS-21.

Four vaccinations were administered intradermally into extremities withintact lymphatic drainage at two-week intervals, followed by twoadditional vaccinations at eight week intervals. Cyclophosphamide(Cytoxan, Mead Johnson and Co., Evansville, Ind.) 200 mg/m2 wasadministered intravenously to all patients 4 to 6 days prior to thefirst vaccination.

Immunological adjuvants: DETOX was produced and supplied by RibiImmunochem Research Inc. (Hamilton, Mont.), formulated as a lyophilizedoil droplet emulsion. It consists of cell wall skeletons (CWS) frombacille Calmette-Guorin and monophosphoryl lipid A (MPLA) fromSalmonella minnesota R595. On the day of vaccination, 0.25 ml DETOX (250μg CWS and 25 μg MPLA) was mixed with the GM2-KLH preparation. Thevaccine (final volume 0.75 ml) was vortexed for 2-3 minutes andadministered to the patients within 15 min. BCG was purchased fromBionetics Research Inc. (Rockville, Md.). On the day of vaccination, 107viable units of BCG in 0.1 ml normal saline were added to the GM2-KLHvaccine in each individual syringe (final volume 0.6 ml). The contentswere mixed and administered to the patients within 15 min. QS-21adjuvant (a homogeneous saponin purified from the bark of Quillajasaponaria Molina) (10, 11) was kindly provided by Cambridge Biotech Inc.(Worcester, Mass.). 100 μg or 200 μg of QS-21 were diluted in 0.25 mlnormal saline and mixed with GM2-KLH. The vaccine (final volume 0.75 ml)was vortexed for 2-3 minutes and administered within 15 min.

Gangliosides

GM2 and GD1b from bovine brain were a gift from Fidia ResearchLaboratory (Abano Terme, Italy). GM3, GM1 and GD1a from bovine brainwere purchased from Sigma Chemical Co. (St. Louis, Mo.). Asialo-GM2 wasprepared by treatment of GM2 with 0.1M trifluoroacetic acid at 100° C.for 1 h followed by separation on a Sep-Pak C18 reversed-phase column(Waters, Milford, Mass.). GD2 was made from GD1b by treatment withβ-galactosidase (12). GD3 isolated from bovine buttermilk was kindlyprovided by Dr. R. K. Yu (Medical College of Virginia, Richmond, Va.).

Reagents and Monoclonal Antibodies

High-performance thin-layer chromatography (HPTLC) silica gel plateswere obtained from E. Merck (Darmstadt, Germany). 4-chloro-1-naphtholand p-nitrophenyl phosphate disodium were obtained from Sigma. Alkalinephosphatase-conjugated goat anti-human IgM (Kierkegaard and Perry Labs,Gaithersburg, Md.) and mouse anti-human IgG-purified (Southern Biotech,Birmingham, Ala.) followed by alkaline phosphatase-conjugated goatanti-mouse IgG (Southern Biotech) were used for enzyme-linkedimmunosorbent assays (ELISA). Horseradish peroxidase-conjugated goatanti-human IgM or IgG purchased from TAGO (Burlingame, Calif.) were usedfor dot blot immune stain and immune thin layer chromatography (ITLC).Rabbit anti-mouse immunoglobulin conjugated to horseradish peroxidasefor ITLC, and rabbit anti-mouse IgM and IgG conjugated to alkalinephosphatase for ELISA were used with control monoclonal mouseantibodies, and were obtained from Zymed (San Francisco, Calif.).Anti-GM2 mAb 696 was obtained from Kyowa Hakko Kogyo (Tokyo, Japan) andanti-GD3 mAb R24 was generated (13).

Serological Assays

ELISA was performed as previously described (6). To control fornonspecific “stickiness”, immune sera were also tested on plates whichwere processed identically but to which no ganglioside had been added,and the reading was subtracted from the value obtained in the presenceof ganglioside. The titer was defined as the highest dilution yielding acorrected absorbance of 0.1 or greater. Immunostaining of gangliosideswith monoclonal antibodies or human sera was performed after spotting onnitrocellulose strips (14) or separation on HPTLC silica gel glassplates as previously described (3). Plates were developed inchloroform/methanol/water (0.25% CaCl2) 50:40:10 (v/v), and gangliosideswere visualized by staining with resorcinol/HCl reagent or monoclonalantibodies.

Determination of IgG Subclass

Determination of IgG subclass was performed by ELISA usingsubclass-specific secondary mouse anti-human IgG1, IgG2, IgG3, and IgG4mAbs. Secondary mAbs from different suppliers (Table 4) were used.Alkaline phosphatase conjugated to goat anti-mouse IgG (SouthernBiotech, Birmingham, Ala.) was used as third antibody at a dilution of1:200.

Complement-Mediated Cytotoxicity Assays

Complement-mediated cytotoxicity assays were performed by a 4 h 51Crrelease assay. Cells from the GM2-positive melanoma cell line SK-MEL-173served as target cells. 2×106 cells were labelled with 100 μCi Na251CrO4(New England Nuclear, Boston, Mass.) in 10% FCS RPMI for 1 h at 37° C.in a CO2 incubator. The cells were washed twice, and 104 cells/well in96-well round-bottom plates (Corning, New York, N.Y.) were labelled andincubated with 1:5 diluted pre- or post-vaccination serum or with mediumalone for 1 h at 37° C. in a CO2 incubator. The cells were washed andincubated with human complement (Sigma) at a dilution of 1:4 for 4 h at37° C. The plates were spun at 500 g for 5 min, and an aliquot of 125 μlof supernatant of each well was harvested for determination of released51Cr. All assays were performed in triplicate and included control wellsfor maximum release in 1% NP-40 (Sigma) and for spontaneous release inthe absence of complement.

The percentage of specific lysis was calculated as follows:${\%\quad{cytotoxicity}} = {\frac{{{Experimental}\quad{release}} - {{spontaneous}\quad{release}}}{{{Maximum}\quad{release}} - {{spontaneous}\quad{release}}} \times 100}$ResultsVaccine Administration and Side Effects

Twenty four patients were immunized with the GM2-KLH vaccine. Groups ofsix patients each received GM2-KLH with no immunological adjuvant orwith DETOX, BCG or QS-21. No local or systemic toxicity was detectedafter administration of GM2-KLH alone. Vaccines containing DETOXresulted in nodule formation at vaccination sites in four of sixpatients which lasted 2-10 weeks. In four patients these were associatedwith 3-10 cm of erythema and induration but only minimal tenderness. Inone patient it was associated with 25 cm erythema and induration afterone immunization and in a second patient low grade fever and malaise for72 hours after the first immunization. In this patient the DETOX dosewas reduced to 50 μg CWS and 5 μg MPLA for subsequent immunizations. BCGproduced local inflammation and crusting at some point in all patientswhich healed after 2-12 weeks. When this occurred, the dose of BCG wasreduced from 1×107 viable units to a final dose of 3×106 units in fourpatients and 1×106 units in one patient. The sixth patient had a historyof tuberculosis exposure and a positive PPD test. He was thereforestarted at a dose of 1×106 units which was eventually reduced to 1×105units. QS-21 induced mild local erythema and tenderness lasting 24-48hours in all patients at the 100 μg dose. The 200 μg dose of QS-21 wasassociated with local tenderness and inflammation lasting 2-10 daysafter all immunizations as well as mild flu-like symptoms including lowgrade fever (<38.5° C.), headache and myalgia lasting 8-24 hours after 3of the 18 immunizations. No neurological abnormalities or other sideeffects were observed.

Antibody Response to GM2-KLH Conjugate Vaccines

Prior to vaccination, IgG antibodies against GM2 were not found, and IgMantibodies were detected only in three patients, with titers of 1:80 intwo 1:320 in one. The remaining 21 patients showed no GM2 reactivitybefore vaccination. ELISA and dot blot immune stain results with seraobtained before and after immunization are summarized in Table 3. IgMantibody titers after immunization with GM2-KLH, or with GM2-KLH and BCGor GM2-KLH and DETOX, were quite similar (median titer 1:80-1:160). Incontrast, 5 of 6 patients immunizated with GM2-KLH and QS-21 showed IgMantibody titers of 1:1280 or more, significantly higher than the titersin the other groups or in patients previously immunized withunconjugated GM2 and BCG vaccines (6). In addition, immunization withGM2-KLH and QS-21 induced a consistent IgG response for the first timein all patients; only 2 of the other 18 patients receiving GM2-KLHvaccines produced comparable IgG titers.

Sequential IgM and IgG antibody titers against GM2 in patients receivingGM2-KLH and QS-21 are shown in FIG. 6. IgM peak titers were seen afterthe third or fourth vaccination and remained high in most patients forat least 20 weeks. Booster immunizations at week 14 and 22 did notfurther increase IgM titers. IgG titers of 1:160 or higher were seen twoweeks after the fourth vaccination in five of six patients. The titersdecreased to 1:40 or less but rapidly increased again after boostervaccination to the previous levels (median 1:160) and remained at thislevel for more than 11 weeks. The second booster vaccination had noclear effect on antibody titers in most cases. Thus, the response tobooster vaccination showed only one of two characteristics of theclassical secondary immune response. The response occurred more rapidly,but antibody titers did not rise higher than after the initialimmunizations.

KLH antibodies were not detected in pretreatment sera. After vaccinationall patients sera showed reactivity with KLH as indicated in Table 3.The highest titers of IgG antibodies were seen after administration withQS-21, significantly higher than in all other groups, including thenext-best group of patients vaccinated with GM2-KLH and BCG (p<0.005).In the QS-21 group, there was no correlation between the strength of theGM2 response and the KLH response.

Specificity Analysis of GM2 Antibodies

The specificity of ganglioside antibodies detected in the patients' serabefore and after immunization was determined by dot blot immune stainutilizing the ganglioside standards GM3, asialo-GM2, GM2, GD1, GD2, GD3,GD1a and GD1b (FIG. 7). Preimmunization IgM and IgG antibodies from mostpatients showed weak reactivity with asialo-GM2, and some patients alsohad IgM antibodies against GM1 and GD1b. Reactivity with thesegangliosides was not altered by immunization. The only vaccine-inducedchanges were strong reactivity with GM2 and weak reactivity with GD2.Dot blot immune stains were graded as 0, 1+, 2+, or 3+. 3+ reactivity ofIgM antibodies against GM2 was seen in the serum of five of six patientsimmunized with GM2-KLH and QS-21, in one of six patients treated withGM2-KLH and BCG, and in two of six patients treated with GM2-KLH withoutadjuvant or GM2-KLH and DETOX. 3+ reactivity of IgG antibodies was seenin five of six patients immunized with GM2-KLH and QS-21 and in none ofthe patients in the other treatment groups.

Postvaccination sera from patients immunized with GM2-KLH and QS-21 werealso tested by immune thin layer chromatography (FIG. 5A) for reactivitywith GM2 and other gangliosides of a melanoma tissue extract. Mostpatients' sera showed strong IgG and IgM reactivity with GM2 isolatedfrom bovine brain or melanoma. Reactivity was seen also with a slowermigrating band in the melanoma extract, presumably GD2 when comparingthe Rf value with a purified GD2 standard (not shown).

To confirm the GD2 crossreactivity of IgG antibodies, postvaccinationserum from patient No. 2 was preincubated with either GM2 or GD2 beforeperforming the immune stain (FIG. 8B). Reactivity with GM2, and with GD2in the melanoma ganglioside extract, was completely inhibited bypreincubation with GM2. On the other hand, preincubation of the sameserum with GD2 resulted in inhibition of GD2 reactivity only, and didnot change reactivity with GM2. These results suggest the presence oftwo populations of antibodies, one reacting with GM2 alone and anotherwith reactivity for GM2 and GD2.

Subclass Determination of IgG Antibodies

High titer IgG sera from the six patients immunized with GM2-KLH andQS-21 were tested by ELISA using a panel of IgG-subclass specificsecondary antibodies. The results are summarized in Table 4. The IgGantibodies in all six sera were of IgG1 and IgG3 subclass.

Complement-Mediated Cytotoxicity

Effector function of anti GM2 antibodies in the serum of patientsvaccinated with GM2-KLH and QS-21 (diluted 1:5) was tested bycomplement-mediated cytotoxicity assays. As shown in Table 5,post-vaccination sera of all six patients lysed GM2-positive SK-MEL-173melanoma cells in the presence of human complement. Prevaccination serashowed no cytotoxicity with addition of complement, and postvaccinationsera were not cytotoxic with GM2 negative melanoma cells or whencomplement was not added. Clearly, these are only preliminary results,and more detailed study of cell surface binding and cytotoxic effectorfunctions of vaccine-induced antibodies and their subclasses is nowunderway.

Discussion

In a series of studies in patients with malignant melanoma, theobjective has been to construct vaccines that are effective in inducingproduction of antibodies against three gangliosides often overexpressedin melanoma—GM2, GD2 and GD3. The initial approach was to vaccinatepatients with unconjugated gangliosides adsorbed to BCG. In this way wewere able to induce antibody production against GM2 (5, 6) but not GD2or GD3. GM2 antibodies induced by GM2-BCG vaccines were mostly of theIgM class, the antibody response was of short duration, and boosterimmunization resulted again in a brief period of IgM antibody productionsimilar to the primary response—all characteristics of a T-cellindependent immune response, well known from studies of othercarbohydrate antigens. Even so, vaccine-induced production of GM2antibodies by patients with Stage III melanoma after surgery wasassociated with increased survival (6, 7). This observation suggestedthat melanoma gangliosides are appropriate candidates for vaccineconstruction, and that melanoma ganglioside vaccines of increasedimmunogenicity might result in superior clinical outcomes. As therelevant epitopes of melanoma gangliosides are carbohydrates it ishelpful to consider studies that have been aimed at increasing theimmunogenicity of other carbohydrate vaccines, notably vaccines againstcertain bacterial infections.

The major distinction of the immune response to carbohydrate antigens,as opposed to protein antigens, is that it does not depend on thethymus. The concept that carbohydrate antigens are thymus-independent(TI) is based on the observation that neonatally thymectomized mice aswell as thymic mice show unimpaired humoral immune responses tobacterial polysaccharides (15). B-cells that respond to TI antigens showseveral characteristic features. They appear later in ontogeny, arelong-lived, and do not require T-cells for activation, at least not invivo. Although T-cells are required for B-cells to respond to TIantigens in vitro, the nature of the T-cell effect is poorly understoodand clearly different from the MHC-restricted T-cell help in theT-dependent antibody response to protein antigens. While T-cells are notindispensable for the in vivo antibody response to TI antigens, antibodylevels are higher when T-cells are present, suggesting a generalaugmenting activity of T-cells, again by unknown mechanisms (16).

A large variety of approaches has been explored in attempts to increasethe immunogenicity of carbohydrate antigens. They include chemicalmodification (17), administration with adjuvants, non-covalentcomplexing with proteins, covalent attachment to immunogenic proteincarriers (18), and replacement of the carbohydrate epitope by a proteinreplica, either peptides synthesized de nova (so-called mimotopes, 19)or anti-idiotypic antibodies (20). Most of these approaches result inincreased T cell help for the carbohydrate specific antibody response.While each has shown promise in initial experimentation, covalentattachment of carbohydrate antigens to immunogenic T-dependent proteincarriers, as first suggested for haptens (21) and then disaccharides(22), is the concept that has been pursued most vigorously, resulting invaccines that have in some instances been shown to be highly effectivein recent clinical trials.

Excellent examples are H. influenzae type b (Hib) polysaccharide proteinconjugate vaccines. Four vaccines that have been developed over the lastdecade differ in the carbohydrate components, protein carriers andlinkers between carbohydrate and protein (23-27). In comparative studiesin children, conjugate vaccines induced a much stronger antibodyresponse than unconjugated Hibphosphoribosyl/ribitolphosphatepolysaccharide (PRP) vaccine (28). Ofparticular interest are observations that young children first immunizedwith HbOC (oligosaccharide-nontoxic diphtheria toxin) or PRP-OMPC (outermembrane protein complex of Neisseria meningitidis type B) vaccines andlater challenged with unconjugated PRP vaccine showed an anamnestic IgGresponse even if challenged at an age at which they do not respond toprimary immunization with the unconjugated vaccine (29, 30). How T-cellsare engaged, and how they interact with Hib PRP-responsive B-cells, isstill far from clear. The fact that increased immunogenicity andT-dependence require a covalent bond between PRP and protein suggeststhat the proximity between protein and PRP must not be disturbed, atleast not in the early phase of antigen processing. As the isotype andbiological activities of antibodies induced by Hib PRP and Hib PRPconjugates are the same, it appears that the B-cells that respond to theconjugate-induced T-cell signal are qualitatively identical with thoseengaged by Hib PRP alone.

Drawing on the substantial experience that has accumulated in thedevelopment of carbohydrate vaccines for bacterial infections,applicants have explored, over the past several years, similarapproaches in attempts to increase the immunogenicity of melanomagangliosides. Chemical modification of GD3, resulting in amide, lactoneor gangliosidol formation, or O-acetylation, produced derivatives thatwere highly effective in inducing antibody production in patients withmelanoma. However, the antibodies induced by these GD3 derivatives didnot cross-react with GD3 (31, 32). An anti-idiotypic antibody BEC-2,mimicking GD3, has been developed by immunizing mice with the monoclonalantibody R24 which recognizes GD3. Rabbits immunized with BEC-2 producedanti-GD3 antibodies (33), and initial studies of the immunogenicity ofBEC-2 in human patients are underway.

Regarding conjugate vaccines, initial studies with GD3 in the mouse wereconcerned with three issues—development of the conjugation method,selection of the carrier protein, and choice of the adjuvant (7). Theoptimal conjugation procedure involved ozone cleavage of the double bondof GD3 in the ceramide backbone, introduction of an aldehyde group, andcoupling to protein aminolysyl groups by reductive amination. Of fivecarriers tested—poly-1-lysine, keyhole limpet hemocyanin (KLH),cationized bovine serum albumin, Neisseria meningitidis outer membraneprotein complex (OMPC), and multiple antigenic peptide constructscontaining four repeats of a malarial T-cell epitope on a branchingpolylysine core—, KLH was found to be most effective. NoncovalentGD3/KLH complexes were not immunogenic. The best adjuvant was QS-21, ahomogeneous saponin fraction purified from the bark of Quillajasaponaria Molina. The characteristics of the antibody response toimmunization with GD3-KLH conjugate and QS-21 included a) a high initialIgM antibody titer, b) a rapid secondary rise of IgM antibody titersafter booster immunizations, c) maintenance of IgM antibody titers afterbooster immunization for up to ten weeks, and d) consistent productionof IgG antibodies at high titers, parallel to IgM antibody productionexcept for the initial delay of two weeks. These findings have now beenreproduced in human melanoma patients by immunization with anotherganglioside conjugate vaccine, GM2-KLH, using the same conjugationprocedure. As in the mouse studies, QS-21 proved to be a significantlymore effective adjuvant than DETOX or BCG, with acceptable toxicity.

The GM2 antibody response had many characteristics of a T cell dependentresponse. It was long-lasting, and antibodies of IgG1 and IgG3 subclass(usually associated with a T cell dependent immune response) wereinduced. As seen with the Hib-PRP vaccines, these isotypes were the sameas those induced occasionally at low titers with unconjugated GM2/BCGvaccines. The lack of a clear booster effect in the sustained high-titerIgM and IgG response after vaccinations three and five months followingthe initial series may be explained by the fact that the patients wereimmunized at two-week intervals initially. In the classical experimentshowing the secondary response to protein antigens, the second injectionof antigen is given four weeks after the first. Antibody levels afterthe first immunization are higher between one and two weeks after theinjection, and then decline to very low levels before the boosterinjection is given after four weeks. In the immunization scheduleapplicants chose, the initial antibody response did not subside butincreased in a stepwise fashion in response to the first fourvaccinations at two-week intervals, anticipating the secondary responsethat is seen in a more dramatic fashion in the classical experiment.Unlike the antibody response to most protein antigens, the IgM responsewas long-lasting, and IgM antibodies remained at higher titer than IgGantibodies, even after repeated booster immunizations, as ischaracteristic for carbohydrate antigens. Hence the immune responseagainst gangliosides which contain a comparably short oligosaccharidechain linked to a lipid backbone and which are autoantigens show much incommon with the immune response against Hib-PRP and other bacterialcarbohydrates.

The development of the GM2-conjugate vaccine will make it possible todetermine whether higher levels of IgM and IgG antibodies against GM2,sustained over longer periods, will be more effective in delayingrecurrence of melanoma than the lower levels of mostly IgM antibodies,present for shorter periods, in patients immunized with unconjugatedGM2. In addition, applicants can now test whether conjugation withimmunogenic protein carriers also confers immunogenicity to GD3 and GD2,major gangliosides which have not induced an antibody response inmelanoma patients when given as unconjugated vaccines. If this can beaccomplished, construction and testing of a polyvalent melanomaganglioside vaccine would be an attractive next step. TABLE 3Serological response of patients receiving GM2-KLH conjugate vaccineswith or without adjuvants in comparison to vaccine containing GM2adherent to BCG (GM2/BCG) Reciprocal GM2 antibody titers after beforeimmunization No. of immunization (Peak) Vaccine Patients IgM IgG IgM IgGGM2-KLH 6 20, 0(5) 0(6) 160(2), 80 160, 0(5) (2), 40, 0 median  0 0 80 0 titers: GM2-KLH + 6 20(2), 0 0(6) 320, 160 160, 0(5) DETOX (4) (4), 0median 10 0 160  0 titers: GM2-KLH + 6 80(2), 20 0(6) 1280, 320 320, 20BCG (2), 0(2) (2), 80 (2), 0(3) (2), 0 median 20 0 200  10 titers:GM2-KLH + QS- 6 320, 20 0(6) 5120(2), 1280, 640 21 (2), 0(3) 1280(3),(2), 320, 320 160, 80 median 10 0 1280  480 titers: GM2/BCG^(a) 58 160,40, 20 0(58) 640(11), 640, 160, (10), 10 320(9), 80(4), (11), 160(15),20, 0(35) 80(8), 10(3), 40(8), 0(48) 20(2), 10(3), 0(2) median  0 0 160 0 titers: Reciprocal KLH antibody stain titers after Dot blot immuneimmunization No. of for GM2 antibodies (Peak) Vaccine Patients IgM IgGIgG GM2-KLH 6 3⁺(2), 2⁺(2), 1⁺(2), 0 540(2), 180, 60 0(2) (4) (3) median 120 titers: GM2-KLH + 6 3⁺(2), 2⁺(3), 2⁺, 0(5) 1620, 540(2), 180 DETOX1⁺ (3) median  360 titers: GM2-KLH + 6 3⁺, 2⁺(3), 0 1⁺(2), 0 4860,1620(2), BCG (2) (4) 540(3) median 1080 titers: GM2-KLH + QS- 6 3⁺(5),2⁺ 3⁺(5), 2⁺ 10240, 5120(2), 21 2560(3) median 3840 titers: GM2/BCG^(a)58 3⁺(32), 2⁺ 3⁺(2), 2⁺ ND (15), 1⁺(4), 0 (5), 1⁺ (7) (4), 0(47) medianND titers:^(a)historical data(6)

TABLE 4 Characterization by IgG-subclass Specific MoAbs of IgG Absinduced against GM2 induced with GM2-KLH plus QS-21 vaccine IgG subclassmABS Reciprocal ELISA Titer against GM2 conc. mAbs Patient No.Specificity (ug/ul) source 1 2 3 4 5 6 IgG 10 SBA^(a) 640 640 640 640640 640 20 BS 40 20 20 nd 20 20 5 ZLI 640 640 640 640 640 640 IgG1 10SBA 10 10 10 10 10 10 20 BS 10 10 0 10 0 0 2 ZLI 10 0 10 0 10 0 IgG2 10SBA 10 0 0 0 0 0 20 BS 0 0 0 0 0 0 2 ZLI 0 0 0 0 0 0 IgG3 10 SBA 20 2020 20 20 20 20 BS 40 80 40 40 40 40 2 ZLI 20 40 20 10 10 20 IgG4 10 SBA0 0 0 0 0 0 20 BS 0 0 0 0 0 0 2 ZLI 0 0 0 0 0 0^(a)SBA, Southern Biotechnology Associates (Birmingham, AL); BS, TheBinding Site Ltd.; ZLI, Zymed Laboratories Inc. (San Francisco, CA)

TABLE 5 Complement lysis of melanoma cell line SK-MEL-178 mediated byGM2 antibodies in sera from patients immunized with GM2-KLH plus QS-21Percentage of lysis^(a) Pre Post Post vaccination vaccinationvaccination Patient serum with serum with serum without No. complementcomplement complement 1 1.3 38.75 3.1 2 2.2 16.9 2.72 3 1.1 14.0 0.9 41.2 26.0 2.2 5 2.1 34.9 1.65 6 10.5 44.7 2.3^(a)Target cells were labeled with ⁵¹Cr and treated with 1:5 dilutedanti sera.

Fourth Series of Experiments

Increasing doses of saponin fraction QS-21 were administered asimmunological adjuvant in a Phase I trial with a constant dose of themelanoma ganglioside GM2 covalently attached to keyhole limpethemocyanin (KLH). Twenty-eight patients with AJCC Stage III or IVmelanoma who were free of disease after surgery were treated with sixvaccinations administered subcutaneously over a 5 month period. Localand systemic reactions were QS-21 dose related. Doses of 100 ug or lessinduced mild local tenderness and inflammation at vaccination siteslasting 2-4 days and occasional brief low grade fever and malaise, butno significant incapacitation. The 200 ug dose induced low grade feverand malaise after 30% of vaccinations and local reactions as large as 20cm in diameter were seen in all patients resulting in restricted usageof the injected extremity for 5-10 days. The titers of IgM and IgGantibodies against GM2, and IgG antibodies against KLH, were highest atthe 100 and 200 ug QS-21 doses. No antibodies against QS-21 weredetected. This trial identifies the 100 ug dose of QS-21 as the optimalwell tolerated dose for induction of antibodies against GM2 and KLH inmelanoma patients.

Introduction

Applicants and others have vaccinated melanoma patients with a series ofwhole melanoma cell vaccines selected for expression of a variety ofglycoprotein and ganglioside antigens (1, 2). The antigen recognizedmost frequently by antibodies in post vaccination sera was theganglioside GM2. GM2 ganglioside is a differentiation antigenoverexpressed on the cell surface of human malignant melanomas. GM2injected alone was found not to be immunogenic and so GM2 vaccinescontaining a variety of immunological adjuvants were tested (3, 4).Immunization with purified GM2 ganglioside adherent to BacillusCalmette-Guérin (BCG) induced production of IgM antibody against GM2 inmost melanoma patients and patients producing antibody had a longerdisease-free interval and survival when compared to patients who did notproduce antibody (4, 5). In a randomized study with 122 melanomapatients who were disease free after surgery, the majority of patients(86%) receiving the GM2/BCG vaccine produced antibodies (6). Patientsthat produced anti-GM2 antibodies had a significantly longer diseasefree interval and overall survival than antibody negative patients.Comparing the two arms of the trial, patients receiving the GM2/BCGvaccine had an 18% improvement in disease free interval and an 11%improvement in survival when compared to the BCG control group, thoughneither difference was statistically significant. The immune responsewas of short duration, mostly IgM and of moderate titer. Similarapproaches with other melanoma gangliosides GD2, GD3 and 9-O-acetylGD3/BCG vaccines in patients resulted in only occasional low titerantibody responses (7). The need for a more effective vaccine wasevident.

Landsteiner's classical experiments with hapten carrier conjugates hadbeen successfully applied to vaccination against a variety of antigensincluding bacterial capsular polysaccharides in infants (8). Based onthis background, several different carbohydrate tumor antigens wereconjugated to keyhole limpet hemocyanin (KLH), and shown to elicithigher titer IgM and IgG antibody responses than previously seen withnonconjugate vaccines (9-11). Applicants tested the immunogenicity ofGD3 linked to a variety of carrier molecules in the mouse and identifiedKLH as the optimal carrier (12). A GM2-KLH conjugate vaccine was testedalone or mixed with immunological adjuvants BCG and DETOX in melanomapatients but the immune response was not significantly different thanthat induced by GM2-BCG vaccines, moderate titer IgM antibodies alonewere induced (13). A more potent immunological adjuvant was required.

QS-21 is a saponin fraction purified to near homogeneity from Quillajasaponaria Molina bark and selected for high adjuvant effect and lowtoxicity (14, 15). Saponins have been used as immunological adjuvants ina variety of settings (reviewed in 15). QS-21 has been used to augmentthe antibody response against an E. coli polysaccharide (16), and theantibody and T cell responses against an HIV-1 envelope protein (17),ovalbumin (18), and feline leukemia virus (19). Cytotoxic T cells havebeen induced against HIV infected cells and ovalbumin transfected cellsby immunization with protein/QS-21 mixtures (17, 18). Applicants testeda variety of new immunological adjuvants with various carbohydrateantigen-KLH conjugate vaccines and the immunological adjuvants QS-21 andSAF_(m) were found to most effectively augment IgM and IgG antibodytiters (9). Experiments focused on QS-21 since it was the least toxicand was available for testing with conjugate vaccines. GD3ganglioside-KLH plus QS-21 vaccines in the mouse induced consistent hightiter IgM and IgG antibody responses (12), providing the basis fortesting this approach in melanoma patients.

Described herein are the results of a Phase I trial using a constantdose of GM2-KLH plus increasing doses of QS-21. Since purified QS-21 orpartially purified saponins such as Quil A had not previously been usedin humans, the goal was to determine the maximum tolerated doseconsistent with repeated outpatient administration and the optimal dosefor augmenting the antibody response to GM2.

Materials and Methods

Gangliosides

GM2 and GD1b from bovine brain, were a gift from Fidia ResearchLaboratory (Abano Terme, Italy). GM3, GM1 and GD1a from bovine brainwere purchased from Sigma Chemical Co. (St. Louis, Mo.). GD2 was madefrom GD1b by treatment with β-galactosidase (20). GD3 was isolated frombovine buttermilk and kindly provided by Dr. R. K. Yu (Medical Collegeof Virginia, Richmond, Va.).

Reagents and Monoclonal Antibodies

HPTLC silica gel plates were obtained from E. Merck (Darmstadt,Germany); 4-chloro-1-naphthol, p-nitrophenyl phosphate disodium fromSigma. Alkaline phosphatase-conjugated goat anti-human IgM (Kierkegaardand Perry Labs, Gaithersburg, Md.) and mouse anti-human IgG-purified(Southern Biotech, Birmingham, Ala.) followed by alkalinephosphatase-conjugated goat anti-mouse IgG (Southern Biotech) were usedfor ELISA. Horseradish peroxidase-conjugated goat anti-human IgM or IgGpurchased from TAGO (Burlingame, Calif.) were used for dot blot immunestain and immune thin layer chromatography. Rabbit anti-mouseimmunoglobulins conjugated to horseradish peroxidase for ITLC, andrabbit anti-mouse IgM and IgG conjugated to alkaline phosphatase forELISA were used with control monoclonal antibodies, and were obtainedfrom Zymed (San Francisco, Calif.). Anti-GM2 mAb 696 was obtained fromKyowa Hakko Kogyo (Tokyo, Japan) and anti-GD3 mAb R24 was generated(21).

Patients

Melanoma patients with AJCC Stage III or IV disease were consideredeligible if all evidence of metastatic disease had been rejected withinthe last 8 months. None of the patients had received prior chemotherapyor radiation therapy. Patients were evaluated for toxicity at the timeof each vaccination. In addition, patients were instructed to measurelocal reactions and to take their temperature. These results were calledin and then reviewed at the next clinic visit. Four patients (one ateach of the 4 dose levels) received only 4 immunizations due to diseaseprogression necessitating other treatment.

Vaccine Preparation and Administration

GM2-KLH conjugate was prepared by Biomira Inc. (Edmonton, Alberta). TheGM2/KLH molar ratio was approximately 800/1 and was supplied at aconcentration of 0.57 mg conjugate per 0.5 ml normal saline. Thisrepresented one patient dose and contained 70 ug GM2, 500 ug KLH and 0.5ml normal saline. QS-21 adjuvant, containing a saponin componentisolated from Quillaja saponaria Molina tree bark, was provided byCambridge Biotech Corp. (Worcester, Mass.). The purity of the QS-21 wasdetermined to be ≧98% by analytical reverse phase HPLC. Ten, 50, 100 or200 ug of QS-21 were diluted in 0.25 ml normal saline and mixed withGM2-KLH. The vaccine (final volume 0.75 ml) was vortexed for 2-3 minutesand administered within 15 min. Four vaccinations were administeredsubcutaneously at two week intervals followed by two more at eight weekintervals. Initially 12 patients were treated, 3 at each of the 4 QS-21doses. Subsequently 1 additional patient was treated at the 10 ug doseand 3 additional patients were treated at the 3 higher doses.Cyclophosphamide (Cytoxan, Mead Johnson and Co., Evansville, Ind.) 200mg/M² was administered IV to all patients 3-6 days prior to the firstvaccination.

Serological and Delayed-Type Hypersensitivity (DTH) Assays

Enzyme-linked Immunosorbent Assays (ELISA) were performed as previouslydescribed for gangliosides (5). For ELISA assays on QS-21,QS-21:ethylenediamine (prepared by linkage of ethylene diamine to theQS-21 glucuronic acid carboxyl group) was plated on glutaraldehydetreated Immulon 4 plates (Dynatech Labs, Chantilly, Va.). To controlnonspecific “stickiness”, immune sera were also tested on plates whichwere processed identically but to which no ganglioside KLH or QS-21 hadbeen added, and the reading was subtracted from the value obtained inthe presence of antigen. The titer was defined as the highest dilutionyielding a corrected absorbance of 0.1 or greater. Immunostaining ofgangliosides with monoclonal antibodies or human sera was performedafter spotting on nitrocellulose strips as previously described (22).Patients were skin tested with GM2, GM2-HSA and KLH at the time of thefifth vaccination. Twenty-five micrograms of each were diluted in 0.05ml PBS and administered intradermally. Results were interpreted asdescribed previously (5).

Results

Toxicity

The number of vaccinations administered containing GM2-KLH alone or withvarious doses of QS-21 and the local and systemic reactions associatedwith these vaccinations is shown in Table 6. While GM2-KLH alone or with10 ug of QS-21 resulted in occasional mild local erythema andinduration, increasing doses of QS-21 resulted in increasing frequencyand severity of local reactions. At the 10 and 50 ug doses, this wasassociated with slight tenderness and 2-4 cm of erythema and indurationlasting 24-48 hours (no toxicity greater than grade 1 was detected). Atthe higher doses these reactions became more prominent with mostreactions as large as 8-10 cm and some (at the 200 ug dose) extending to20 cm in diameter. These reactions generally lasted 2-4 days and in onecase as long as 7 days at the 100 ug dose, and generally lasted 7 daysand in one case as long as 10 days at the 200 ug dose. In no case wereanalgesics more potent than tylenol required and patients at the 10, 50and 100 ug doses continued to pursue their normal activities. Use of thevaccinated extremity was restricted for 5-8 days after administration ofthe majority of vaccines containing 200 ug QS-21, but all evidence oflocal reactions had disappeared by two weeks when the patients were nextexamined. No ulceration and drainage or subcutaneous nodules (as seenwith most other adjuvants) were detectable. While occasional brief,mild, low grade fevers or myalgias were seen after vaccinationscontaining the lower 3 doses of QS-21, approximately one third of thevaccinations containing 200 ug of QS-21 were associated with thesesymptoms. In general, systemic symptoms were most prominent after thesecond immunization. All patients in the 200 ug QS-21 group were taperedto 100 ug or 50 ug QS-21 for their third and fourth immunizations due toconcern over the increasing local and systemic reactions (side effectsfor these tapered dose immunizations are listed under 100 ug and 50 ugin Table 6). These doses were well tolerated and so all 5 patients (onepatient had disease progression and was taken off protocol after 4immunizations) were increased back to 200 ug for the fifth immunization.Two of the five fifth immunizations were associated with fever andflu-like symptoms, one of these was also associated with 20 cm of localerythema and induration. This patient's QS-21 dose was decreased to 100ug in the sixth vaccination, no systemic symptoms developed and thelocal reaction was mild (3-4 cm erythema and induration). None of thefour remaining sixth immunizations with 200 ug QS-21 were associatedwith systemic symptoms. While overall the systemic symptoms were quitemild, the local erythema, induration and tenderness were prominentenough with the 200 ug dose that applicants chose not to proceed withhigher doses of QS-21.

Antibody Response Against GM2 after Vaccinations (see Table 7)

Immunization with GM2-KLH alone resulted in antibody titers quitesimilar to those seen in previous studies with vaccines containing GM2adherent to the surface of BCG. Five of 6 patients produced IgMantibody, only 1 patient produced IgG antibody (of low titer). The IgMantibody titers increased with increasing doses of QS-21. Reciprocalmedian IgM antibody titer after immunization with GM2-KLH alone was 80,after 10 or 50 ug of QS-21 was 480 and 240 (respectively), and after 100or 200 ug of QS-21 was 1280. IgG antibody titers increased progressivelywith increasing doses as well, from 0 in patients receiving GM2-KLHalone to 10, 60, 200 and 640 in patients receiving 10, 50, 100 and 200ug of QS-21 respectively. Prominent IgM and detectable IgG antibodies(by ELISA and dot blot immune stains) were seen in every patientreceiving GM2-KLH plus QS-21. Sequential IgM and IgG antibody titers forthe groups of six patients receiving the 100 and 200 ug QS-21 doses areshown in FIG. 9. Overall, the antibody response is quite similar in thetwo groups though IgG ELISA titers were slightly higher with the 200 ugdose. Dot blot immune stains against GM2 confirmed the specificity forGM2 and the similarity of reactions at the 100 and 200 ug doses (seeTable 7 and FIG. 10). The median IgM dot blot reaction against GM2increased from 2+ in patients receiving GM2-KLH alone to between 2+ and3+ in patients receiving 10 or 50 ug of QS-21 to 3+ in patientsreceiving 100 or 200 ug of QS-21. IgG reactions increased from 0 inpatients receiving no QS-21 to 2+ in patients receiving 10 or 50 ugQS-21, to 3+ in patients receiving 100 or 200 ug QS-21.

Specificity Analysis of GM2 Antibody Responses

Dot blot immune stains with sera obtained prior to and afterimmunizations for 2 representative patients in each of the 5 treatmentgroups are shown in FIG. 10. Prior to immunization, IgM reactivityagainst GM1 was demonstrated in sera of many patients as applicants havepreviously described, but no reactivity was detected against GM2 orother gangliosides. Following immunization the GM1 reactivity was noteffected but IgM and IgG antibodies against GM2 were seen in allpatients. As applicants have previously described (13), these inducedGM2 antibodies sometimes showed cross-reactivity with GD2 (patient 1,100 ug dose).

Serological Responses Against KLH and OS-21

As shown in Table 7, reactivity against KLH was not seen prior toimmunization and increased progressively with increasing doses of QS-21.While the 50, 100 and 200 ug doses of QS-21 induced significantly highertiters of IgG antibodies against KLH than the 0 or 10 ug dose, there wasno significant difference between the serological titers induced at the50, 100 and 200 ug doses. Antibody titers to QS-21 were assayed by ELISAafter four immunizations. There was no significant increase in antibodyto QS-21 between pre- and post-immunization serum samples (data notshown).

DTH Responses to GM2 and KLH

No erythema or induration were detected at GM2 or GM2-HSA skin testsites. Prominent erythematous reactions were detected at 24 and 48 hoursat most KLH skin test sites and these reactions were largest in patientsreceiving the 200 ug dose (median diameters 4.0 by 6.0 cm). These wereassociated with only minimal induration, suggesting a combination DTHand antibody mediated reaction, making precise quantification of the DTHresponse (by patients at home) difficult.

Further Clinical Trials

Utilizing the same dose of vaccine as above (Biomira, Inc.) six patientswith advanced disease were treated. An additional six patients withadvanced disease were treated with 1/10 the GM2-KLH dose again with 100μg QS-21. Antibody titers in both groups were lower than seen inpatients with early disease but the 7 μg GM2 dose appeared equally aseffective at inducing IgM and IgG antibody titers as the 70 μg dose.

In another study, 6 patients with early disease were treated with aGM2-KLH construct. IgG antibody titers were comparable to those seenafter administration of the Biomira construct though the IgM antibodytiters appeared somewhat lower. However, all patients made antibodyfollowing immunization and the vaccine was well tolerated.

A further study will involve 18 patients receiving vaccinations ofGM2-KLH, initially at weekly intervals (a schedule shown to be superiorin mouse studies). The patients are randomized, some receiving nopretreatment with low dose cyclophosphamide, and the others receivingpretreatment with low dose cyclophosphamide. At this time 10 patientshave been accrued and there appears to be no difference between patientsreceiving cyclophosphamide and those not receiving cyclophosphamide, andthe antibody titers with the new schedule appear at this time to besuperior to those in previous trials with the same conjugate.

Discussion

This was a Phase I trial designed to identify the maximum tolerated dose(MTD) of QS-21 for use in the outpatient setting and the dose of QS-21providing the greatest immune potentiating effect. The probable MTD wasidentified as 200 ug QS-21 per vaccination. While the low grade feversand malaise, and the large local inflammatory reactions at the 200 ugdose, cannot be considered the MTD in the context of high dose IL2 orcombination chemotherapy as utilized in hospitalized patients withadvanced melanoma (23, 24), they were significant in the context ofoutpatient treatment of cancer patients in the adjuvant setting, andwould be unacceptable for immunization against infectious diseases innormal recipients. The 100 ug dose, however, resulted in only 2 episodesof low grade fever in 44 injections and the local inflammatory responsesdid not interfere with daily activities and were generally limited to2-4 days. This dose was well tolerated in the patient population of theclinical trial. There was a clear increase in local and systemictoxicity with progression from the 100 ug dose to 200 ug which made ushesitant to continue dose escalations. While fever and malaise at thehighest dose were similar to those sometimes seen with otherimmunological adjuvants such as BCG and DETOX (15, 25), local reactionswere quite different. The 200 ug dose of QS-21 injected subcutaneouslyresulted in a 10-20 centimeter diameter area of erythema and indurationwhich was hot and tender to the touch. This local response is morediffuse than the response generally seen with doses of DETOX or BCGinducing comparable systemic symptoms. A surprising feature of theseresponses was that several days later (or at most 10 days later) thesereactions had completely abated and there was no evidence that thevaccination had been administered to that sight. No ulceration, drainageor nodule formation at the injection sight was detected in any patient.

A second surprising finding in this study was that QS-21 at any of thedoses used resulted in a qualitatively different immune response to GM2ganglioside. Even at the 10 ug dose all patients produced IgG antibodiesdetectable by dot blot immune stains against GM2. GM2-KLH vaccines aloneor with optimal doses of BCG or DETOX, or GM2 adherent to the surface ofBCG, salmonella Minnesota mutant R595 or proteosomes, had only rarelyresulted in more than 1 detectable IgG response per 6 immunized patients(2, 4, 5). IgM and IgG antibody titers against GM2 continued to increasewith increasing doses of QS-21 but appeared to reach a plateau betweenthe 100 and 200 ug dose. IgG antibodies against KLH likewise increasedwith increasing doses of QS-21. Peak IgG titers were significantlyhigher at the 50, 100 and 200 ug doses than at the 0 or 10 ug doses, butthe titers at the three higher doses were not significantly differentfrom each other. Results demonstrate that the 100 and 200 ug doses ofQS-21 induce the optimal antibody response against GM2 and that the 100ug dose is better tolerated. The primarily erythematous skin testresponses against KLH in patients immunized with GM2-KLH alone orGM2-KLH plus various doses of QS-21 was unexpected. It may represent acombination of antibody mediated arthus-like and DTH responses. It isalso possible that the lower dose of KLH in the skin tests than thatused in studies by others (26) (25 ug as opposed to 100 ug) wasinsufficient to produce a strong detectable DTH reaction, or that thehigh epitope density of GM2 ganglioside on the KLH in the vaccines didnot permit the normal antigen processing and presentation required forinduction of a classical DTH response against KLH.

Despite the wide spread use of QS-21 and other saponin containingadjuvants in experimental animals and veterinary practice (14-19), theirmechanism of action remains unknown. It is possible to make someinferences on the mechanism of action of QS-21 from these studies. Thelack of palpable nodules at injection sights suggest that a depo effectwith granuloma formation does not occur. Despite the potency of QS-21 asan adjuvant, it appears to be a poor immunogen. Even after repeatedimmunizations, no detectable antibody responses resulted. So unlikeother adjuvants such as C parvum, BCG and complete Freund's adjuvant,the immunogenicity of QS-21 may not contribute to its adjuvant effect.Low grade fever and malaise seen at the 200 ug dose, and diffuseerythema and induration at injection sights, suggest that cytokinerelease is involved. Intradermal injection of IL-2 has resulted in verysimilar reactions which on biopsy are characteristic of classical DTHresponses (27, 28). Partial switching of GM2 antibodies to an IgGresponse suggests induction of T cell help, perhaps as a consequence ofthis cytokine induction.

Applicants have recently demonstrated the superiority of KLH over othercarriers tested for augmentation of ganglioside immunogenicity (12) andapplicants demonstrate here the optimal dose of QS-21 for furtheraugmentation of this effect. As applicants prepare for a large phase IIItrial of ganglioside vaccine in AJCC stage III melanoma patients, thereare a number of variables which remain to be tested. Applicants arecurrently conducting preclinical studies with this ganglioside-KLH plusQS-21 vaccine plus the use of several other immunological adjuvants. Inaddition, clinical trials with different schedules of immunization anddifferent doses of GM2-KLH are planned. The surprising findings in thephase I trial described here are 1) no evidence of granuloma formationwas detected at any dose of QS-21 and 2) that at the well tolerated doseof 100 ug, the serological response against GM2 was strikingly superior,quantitatively and qualitatively, to any seen with previously tested GM2vaccines. TABLE 6 NUMBER OF ADVERSE REACTIONS OCCURRING (AND THEIRSEVERITY) AT EACH QS-21 DOSE IN PATIENTS RECEIVING VACCINES CONTAINING500 UG GM2-KLH ± QS-21 Dose of QS-21 and Adverse Reactions 0 10 ug 50 ug100 ug 200 ug Number of Vaccinations ADVERSE REACTIONS: 35 22 34 44 23Local Tenderness and Pain 6(1)  7(1)* 21(1)  34(2) 23(2)  Erythema andInduration 6(1) 4(1) 17(1)  24(2) 21(3)  Systemic Fever 0 0 3(1)  2(1)7(1) Headache 0 0 0 0 1(1) Myalgia 0 1(1) 2(1) 0 6(1) Nausea or Vomiting0 1(1) 1(1) 0 1(1)*7(1): Local pain or tenderness was experienced after 7 of 22vaccinations containing 10 ug QS-21.The number in parenthesis indicates median severity by the NCI CommonToxicity Criteria, in this case mild local tenderness.For local toxicity, (1) Minimal tenderness and erythema, (2) Moderateerythema and induration <10 cm, pain not requiring narcotic analgesics,(3) Erythema and induration ≧10 cm, pain requiring narcotic analgesics(not seen).For systemic toxicity, (1) refers to temperatures 101°-103° F., mildheadaches, mild muscle aches or mild nausea with no vomiting.

TABLE 7 SEROLOGIC RESPONSE AFTER VACCINATION WITH GM2-KLH PLUS VARIOUSDOSES OF QS-21 PEAK ELISA TITER AGAINST GM2 VACCINE # OF PretreatmentAfter Treatment GM2-KLH PLUS PATIENTS IgM IgG IgM IgG No adjuvant 6 20,0(5)* 0(6) 160(2), 80(2), 40, 0(5). 40, 0  10 μg QS-21 4 0(4) 0(4) 1280,640, 320, 80 80, 20, 0(2)  50 μg QS-21 6 20, 0(5) 0(6) 640, 320(2),160(3) 80(3), 40(2), 20 100 μg QS-21 6 40, 0(5) 0(6) 5120, 1280(3), 160,80 2560, 640, 320, 80, 40(2) 200 μg QS-21 6 320, 20, 0(6) 5120, 1280(3),320(2) 1280(2), 640(2), 0(4) 320, 80 PEAK DOT BLOT IMMUNE SYSTEM IgGELISA VACCINE # OF RESPONSE AGAINST GM2 TITER AGAINST KLH GM2-KLH PLUSPATIENTS IgM IgG Pretreatment After Treatment No adjuvant 6 3+, 2+(3),1+, 0 1+(2), 0(4) 0(6) 360, 180(3), 20(2)  10 μg QS-21 4 3+(2), 2+(2)2+(3), 1+ 0(4) 810, 270(2), 90  50 μg QS-21 6 3+(3), 2+(3) 3+(2), 2+(2),0(6) 2430(3), 810(2), 90 1+(2) 100 μg QS-21 6 3+(5), 2+ 3+(4), 2+(2)0(6) 21, 870, 2430(3), 810(2) 200 μg QS-21 6 3+(4), 2+(2) 3+(4), 2+(2)0(6) 21, 870, 7290(3), 2430, 810*One patient had a pretreatment peak titer ELISA response of 1/20.The number in parenthesis indicates the number of patients with a givenresponse. In this case, five patients had no detectable GM2 antibodies.

Fifth Series of Experiments

Introduction

The incidence of malignant melanoma has increased rapidly over the lastdecade. In 1992, over 32,000 individuals in the United States developedmelanoma, and 6,700 deaths from melanoma were recorded (1). Followingadequate resection of primary melanoma the five-year survival rateranges from greater than 95% for patients with primary tumors of ≦0.75mm thickness to 50% for those with primary tumors of >4.0 mm thickness(2). Patients with regional lymph node metastases (AJCC Stage III), havea 5 year survival of 25-35% after elective or therapeutic dissection(3). No adjuvant therapy has been shown to reduce the rate of recurrenceand increase survival after surgery in these patients. A large varietyof agents have been tested in these adjuvant trials (reviewed in refs.4, 5) including chemotherapy, nonspecific immune stimulators,interferons, and various types of melanoma vaccines. With regard tomelanoma vaccines, the challenge has been to develop methods that canmonitor immunogenicity of the vaccines in terms of strength andspecificity of the resulting immune response. Because humoral immunereactions to vaccines can be quantitated and analyzed with a precisionthat is only now becoming possible with cellular immune reactions,applicants have focused on melanoma antigens that elicit humoralimmunity. Of these serologically defined antigens, gangliosides,particularly GM2, have emerged as attractive targets for activeimmunization (5-8). Applicants have recently shown that immunizationwith purified GM2 adherent to BCG, after pretreatment with low dose Cy,resulted in the induction of IgM antibodies in a high percentage ofmelanoma patients (8, 9). The GM2 antibody response showed a T-cellindependent pattern, i.e., short duration, lack of an IgG response, andlack of a booster effect of repeated vaccinations. The inducedantibodies were cytotoxic for GM2-expressing melanoma cells in thepresence of human complement, and patients that produced GM2 antibodiesafter immunization showed significantly longer disease-free interval andsurvival than patients that did not (9). The purpose of the presentstudy was to confirm the beneficial effects of vaccine-induced GM2antibody production in a randomized controlled trial.

Materials and Methods

Patients

Patients with pathologically documented melanoma metastases restrictedto regional skin and lymph nodes (AJCC Stage III) were eligible for aperiod of 2 weeks to 13 weeks after complete resection of skinmetastases or regional lymph nodes. Other eligibility criteria included:age >15 years; Karnofsky performance status ≧90; serum creatinine <2.5mg/dl, serum bilirubin <2.5 mg/dl; no other cancers; no chemotherapy orradiation therapy during the 8 week period preceding vaccination.Pregnant women were excluded. All patients were examined, andeligibility determined, within two weeks prior to randomization.Subsequent clinical follow-up was performed by the patient's primaryoncologist.

Randomization and Follow-Up

Informed consent was obtained from all patients and they were stratifiedby number of positive lymph nodes (1, 2-4, ≧5), presence of intransitdisease, and interval between surgery and vaccination (2-6 weeks, 7-13weeks). Patients were randomized by computer (with adaptive allocationof block sizes to ensure reasonably even numbers in the two arms) to bevaccinated with GM2/BCG or with BCG alone. Neither patients nor medicalpersonnel were informed of the type of vaccine administered until twoweeks after the final vaccination. Follow-up information on all patientswas obtained over a one-week period every 4-6 months by telephoningtheir primary physicians. Dates of recurrence documentedradiographically or pathologically were used for determiningdisease-free interval. Since the two nonspecific components of thisvaccination approach, BCG and low-dose cyclophosphamide, may havedemonstratable anti-tumor activity (10, 11, 12), they were consideredmore appropriate than no treatment for the control arm.

GM2/BCG Vaccine

The ganglioside GM2 used for vaccine production was obtained from twosources, brain tissue from cats with Tay-Sachs disease (transmitted inthese cats as an autosomal recessive trait), and GM1 prepared frombrains of domestic cows and purchased from Supelco, Bellefonte, Pa.Slices of Tay-Sachs cat brain (provided by Dr. Mario Ratazzi, Mt. SinaiHospital, New York, N.Y.) were extracted with chloroform/methanol, andthe extract was peracetylated, subjected to florisil chromatography toremove phospholipids, deacetylated, dialyzed and subjected toDEAE-Sephadex column chromatography (13). After dialysis, GM2, the majorfraction, was separated by preparative thin layer chromatography. GM2was prepared from bovine brain GM1 by cleavage of the terminal galactoseusing β-galactosidase, as previously described (14). GM2 from the twosources was indistinguishable by thin layer chromatography (TLC) andimmune TLC using murine monoclonal antibodies recognizing GM2 (13). Eachbatch of GM2 was more than 98% pure as defined by thin layerchromatography and densitometric scanning. Batches were tested bystandard tests for sterility, and for safety in guinea pigs and mice.

Purified GM2 was dried and stored at 4° C. Tice strain BCG (Universityof Illinois, Chicago, Ill.), 10⁷ viable units (or 3×10⁵ viable units foruse in patients with a positive PPD test) was suspended in distilledwater by sonication together with 200 ug of dried purified GM2. Thesuspension was lyophilized and stored at −80° C. The residue wasresuspended in 0.5 ml phosphate-buffered saline (PBS) shortly beforevaccine administration. Under these conditions, GM2 has been found toadhere to BCG, presumably by hydrophobic bonds, as applicants havepreviously reported (8). BCG was suspended in PBS for use in the controlgroup. All patients in the GM2/BCG group received 200 ug GM2 pervaccination. These vaccines and the vaccination protocol were approvedby the Memorial Hospital Institutional Review Board and used under anIND with the U.S. Food and Drug Administration. The initial patientsreceived GM2 of cat brain origin. Subsequently, an increasing proportionof GM1 derived GM2 was used because cat brain GM2 was no longeravailable. All patients received intradermal vaccine injections into6-10 sites of an extremity with intact lymphatic drainage, and this wasrepeated twice at two-week intervals, using different extremities eachtime. Booster immunizations were administered two and five months afterthe initial series of vaccinations.

Cy Administration

A single dose of 200 mg/M² Cy (Cytoxan; Mead Johnson and Co.,Evansville, Ind.) was administered intravenously to all patients five toseven days prior to the first and fourth vaccine injections.

Ganglioside Reagents

GD2 was prepared by treating GD1b with β-galactosidase (G. W. Jourdian,University of Michigan, Ann Arbor, Mich.) according to published methods(14). GM1 was purchased from Supelco (Bellefonte, Pa.). GD3 was a giftfrom Fidia Research Laboratories (Abano Terme, Italy), and GD1b waspurchased from the same source. GM3 was purified from dog erythrocytes(13). GM2 for dot blot immune stains (FIG. 11) was prepared from GM1 bytreatment with β-galactosidase or extracted from human melanoma biopsyspecimens as previously described (13).

Serological Procedures

Sera were obtained at the time of each vaccination, 2 and 6 weeks aftervaccines 3, 4 and 5, and 3 months after vaccine 5. The ELISA for GM2 orBCG antibodies (9) was performed with patient sera and rabbit anti-humanIgM or anti-human IgG second antibody, or Protein A conjugated withalkaline phosphatase (Kirkegaard and Perry Labs, Gaithersburg, Md.).Readings obtained with normal sera from donors without GM2 reactivitywere subtracted from readings obtained with the patients' sera. Antibodytiter was defined as the highest serum dilution yielding a correctedabsorbance of >0.10 as previously described (9). Dot blot immune stainswere performed as previously described (8, 9), and graded 0, 1+, 2+ or3+ as shown in FIG. 11. Sera were categorized as positive if GM2reactivity was 2+ or 3+ by dot blot with ELISA titer ≧1/20 or 1+ by dotblot with an ELISA titer ≧1/80. Complement dependent cytotoxicity assayswere performed as previously described with normal human serum (diluted1/3) as complement source and visual quantification of nonviable cellsby Giemsa stain (8, 15).

Skin Tests for Delayed-Type Hypersensitivity (DTH)

Unilamellar liposomes were prepared from egg phosphatidyl choline andpassed through a 0.1 micron filter ten times. On the day of skintesting, (2 weeks after the fourth vaccination) 1.5 mg phosphatidylcholine liposomes and 25 ug GM2 were mixed in PBS and sonicated in aBranson 1200 water bath sonicator (Shelton, Conn.). Skin tests (0.1 mlvolume) with a) 25 ug GM2 and b) 25 ug/1.5 mg GM2/liposomes wereperformed and read at 48 hours as previously described (16, 17).

Results

Patient Characteristics

A total of 123 patients were enrolled between Mar. 10, 1987 and Mar. 17,1989. On review, one patient was found to have AJCC Stage II disease (nopositive lymph nodes) and was therefore not included in the analysis.All 122 eligible patients, regardless of the number of vaccinesreceived, are included as randomized in the analysis. Two patients (onein each group) decided to leave the study after one immunization becausethey found it too difficult to deal with the uncertainty of experimentaltherapy and randomization. One patient in the BCG group had receivedonly two immunizations when disease progression was detected, and thetreatment was discontinued. All other patients received at least theinitial series of three immunizations and remained on protocol until itscompletion or until disease progression. One patient in the BCG groupaccidentally received GM2/BCG in the fifth vaccination (he produced GM2antibody and remained free of disease). The median follow-up is fiveyears and three months, with a minimum follow-up of four years and threemonths after pre-randomization surgery.

The characteristics of the 122 randomized patients are shown in Table 8.The single most important prognostic indicator for patients with StageIII melanoma is the number of positive lymph nodes (3). Other factorsassociated with a poor prognosis include head and neck or trunk as theprimary site, and intransit melanoma (3). The GM2/BCG and BCG groupswere comparable with regard to these prognostic indicators. TABLE 8PATIENT CHARACTERISTICS TREATMENT GM2 GROUP ANTIBODIES BCG GM2/BCGPositive Negative CHARACTERISTIC No. of Patients 64 58 64 58 Sex (M/F)48/16 37/21 48/16 37/21 Median Age 48.5 46 49.5 44 (Range) (27, 75) (16,74) (31, 75) (16, 74) Time Since Surgery 14-42 days 21 22 20 23 43-91days 43 36 44 35 Primary Site Head and Neck 7 7 8 6 Trunk 29 24 28 25Extremity 23 17 21 19 Not Detected 5 10 7 8 Depth of Primary  <2 mm 1512 15 12 2-4 mm 20 15 19 16  >4 mm 10 11 10 11 Not Available 19 20 20 19Intransit Disease Absent 54 52 54 52 Present 10 6 10 6 Number of LymphNodes with Metastases   1 27 24 26 25 2-4 23 23 24 22 >4 14 11 14 11Side Effects

Pretreatment with Cy occasionally resulted in mild nausea or queasinessfor 2-24 hours but was mostly well tolerated. Intradermal injection ofBCG resulted in inflammation and eschar formation with drainage in allpatients receiving three or more immunizations. To keep the inflammatoryresponse at an acceptable level, the dose of BCG was decreased by afactor of 3 when eschar formation or drainage was first detected.Consequently, the median BCG dose at the time of the final immunizationwas 3×10⁶ viable units in both treatment groups. The dose of 10⁷ unitswas maintained in only 10% of patients, and some patients required afurther dose reduction to 10⁶ units. GM2 had no effect on theinflammatory response to BCG and caused no additional side effects.

Serological Response Against GM2

Sequential IgM anti-GM2 antibody titers before and after vaccinationwith GM2/BCG are shown in FIG. 10 for the first five patients treated(in 1987) and the last five patients treated (in 1989) who received thefull series of GM2/BCG immunizations. As in a previous study (9), theantibody response was predominantly of the IgM type, and frequentlyshowed an increase in titer after each immunization that resembled arepetitive primary response rather than an amnestic secondary response.Pre-immunization and peak titer post-immunization sera from all patientswere also tested by dot blot immune stain to confirm the specificity ofthe antibodies detected by ELISA. Dot blot immune stains with the tensera shown in FIG. 10 are shown in FIG. 11. Occasional low-titerpretreatment reactivity against GM1 was detected as previously described(8, 9). Vaccine induced reactivity was restricted to GM2; no reactivitywas seen with GM3, GD2, GD1b or GD3. GM2 antibodies were able to mediatecomplement dependent cytotoxicity in tests on GM2 expressing tumor cellswith human complement (Table 9). No change in this pattern of antibodyresponse and specificity was seen over the two year period of patientaccrual. The results of serological analysis for all patients aresummarized in Table 10. Seven of the 64 patients in the BCG arm werefound to exhibit GM2 antibodies both by ELISA (median titer 1/40) anddot blot immune stain (median 2+). The antibodies were preexisting in 5cases and were first detected after BCG vaccination in 2 cases. Titersof GM2 antibody increased 4-fold or more after BCG immunization in 3 of5 patients with GM2 antibody prior to BCG vaccination. In contrast, onlyone of the 58 patients receiving GM2/BCG exhibited GM2 reactivity byboth assays before immunization and 50 of 58 patients showed GM2antibodies (median ELISA titer 1/160) in their serum after immunization,confirmed by dot blot immune stain (median titer 3+). The overall IgManti-GM2 serologic response rate (2+ or 3+ dot blot and ELISA titer≧1/20 or 1+by dot blot and ELISA titer ≧1/80) in the two groups was 11%for BCG alone and 86% for GM2/BCG.

IgG antibodies were not detected in any patient's serum prior toimmunization and were not induced in any patient by BCG immunization(Table 10). Vaccination with GM2/BCG induced a positive IgG antibodyresponse in eight patients (median ELISA titer 1/80, median dot blot2+). These responses were short-lived (median duration 8 weeks) and didnot generally increase with additional immunizations. IgG reactivity inall cases was restricted to GM2 (data not shown). All eight patientsalso had IgM antibodies which were of higher titer and longer durationthan the IgG antibodies. TABLE 9 COMPARISON OF GM2 ANTIBODY TITERSBEFORE AND AFTER IMMUNIZATION GM2/BCG, AS DETERMINED BY ELISA ANDCYTOTOXICITY TESTS WITH HUMAN COMPLEMENT O.D. AT 1/40 CYTOTOXICITYTITER* IgM ELISA SERUM 20% LYSIS 50% LYSIS PATIENT TITER DILUTIONEndpoint Endpoint 6  20 0.05  20 0 160 0.412 20 0 7  0 0.066  5 0  800.134  80 5 8  0 0.017  0 0 320 0.78  80 5 10  10 0.057  0 0 160 0.455 80 20 12  0 0.011  5 0 160 0.506  320+ 20 53  0 0.004  20 0  320+ 0.896 320+ 320 54  0 0.000  5 0 160 0.387  20 5 56  0 0.013  20 0 320 0.418320 20 57  0 0.028  80 20 640 0.811 320 80 58  0 0.029  5 0 320 0.718320 20*Target cells: astrocytoma cell line SK-MG 6.

TABLE 10 GM2 ANTIBODIES IN THE SERUM OF MELANOMA PATIENTS BEFORE ANDAFTER IMMUNIZATION WITH GM2/BCG, OR BCG VACCINES NO. OF PATIENTS WITHNO. OF PATIENTS WITH DOT BLOT TOTAL NO. ELISA TITER (RECIPROCAL) IMMUNESTAIN VACCINE PATIENTS 0 10 20 40 80 160 320 640 0 1+ 2+ 3+ IgMANTIBODIES GM2/BCG BEFORE VACCINATION 58 35 11 10 1 1 56 1 1 AFTERVACCINATION 58 2 3 2 8 8 15 9 11 7 4 15 32 BCG BEFORE VACCINATION 64 4010 8 4 2 57 2 5 AFTER VACCINATION 64 26 11 14 7 4 1 1 57 2 3 2 IgGANTIBODIES GM2/BCG BEFORE VACCINATION 58 58 58 AFTER VACCINATION 58 48 31 4 1 1 47 4 5 2 BCG BEFORE VACCINATION 64 64 64 AFTER VACCINATION 64 6464DTH Response to GM2

Skin tests for DTH against GM2 and other antigens were performed afterthe initial series of three vaccinations. All patients receiving threeor more immunizations developed strong DTH reactions to BCG. Eightpatients (four vaccinated with BCG and four vaccinated with GM2/BCG)showed positive DTH reactions to GM2 and GM2 liposomes (used to keep GM2at the skin test site) ranging from 10 to 33 mm of induration. Furthertests revealed that these 8 patients showed similar reactivity withganglioside free liposomes and with other gangliosides. Thus, there wasno evidence for GM2 specific DTH in any patient.

Serologic Response Against BCG

Sera from all patients were tested for IgG ELISA reactivity against 10³viable organisms/well BCG dried onto ELISA plates as described forgangliosides. No reactivity was seen before vaccination.Post-vaccination sera showed BCG antibody titers of 1/40-1/80 in bothtreatment groups, independent of whether or not the patients producedGM2 antibodies.

Correlation of Antibody Response and Immunization with Disease-FreeInterval and Survival

Comparison of disease-free interval and overall survival for allpatients producing GM2 antibodies documented by two assays (ELISA titerof ≧1/180 and dot blot 1+ or ELISA titer ≧1/20 and dot blot ≧2+) withthose of patients who did not produce GM2 antibody is shown in FIG. 12.Significant differences were seen in both disease-free interval andoverall survival (p=0.004 and 0.02 respectively, by log rank test). Ofthe six patients who produced GM2 antibody prior to immunization (fivereceiving BCG and one receiving GM2/BCG), only one (a patient in the BCGgroup) developed recurrent disease, suggesting that the naturalproduction of GM2 antibodies in patients with melanoma is associatedwith a favorable course. Conversely, of the eight patients who did notproduce GM2 antibodies after vaccination with GM2/BCG, six developedrecurrent melanoma and died, suggesting that failure to produce GM2antibodies after vaccination indicates an unfavorable prognosis. MedianBCG antibody titers in the six patients producing natural GM2 antibodieswere the same as the titers in the eight GM2 antibody-negative patientsin the GM2/BCG arm, indicating that these patients did not differ intheir ability to mount a serologic response to an unrelated antigen.

When the six patients producing GM2 antibodies prior to randomization(five in the BCG arm and one in the GM2/BCG arm) were excluded from theanalysis, disease-free interval of the GM2/BCG group was significantlylonger than that of the BCG group (p=0.02 by logrank test), and a trendtoward longer overall survival was also observed for the GM2/BCG group(FIG. 13). The curves plateau at 40-52 months, with differences of 23%and 14% in disease-free interval and overall survival, respectively. Thesmall number of patients producing IgG antibodies reactive with GM2after immunization makes it impossible to draw conclusion about therelative merits of IgM and IgG antibodies against GM2. Five of the eightpatients positive for IgG GM2 antibodies remain free of disease at thistime.

Comparison of the two treatment groups as randomized is shown in FIG.14. The curves plateau at 40 to 52 months with a 18% difference indisease-free interval and 11% difference in overall survival in favor ofvaccination with GM2/BCG. These differences were not statisticallysignificant. Disease-free interval (30%) and overall survival (46%)rates at 51 months (the minimum period of follow-up) in the BCG groupare similar to the rates applicants observed earlier in patientsrandomized to receive BCG or no treatment (28). As shown in FIG. 14, abeneficial effect of immunization was more evident when the twotreatment groups were stratified for number of positive nodes (1 versus2 or more). Immunization had less impact on disease free interval ratesin patients with only one positive node. In contrast, the disease-freeinterval of patients with two or more positive nodes who receivedGM2/BCG was significantly longer than that of patients immunized withBCG alone (p=0.02 by logrank test). A similar trend was seen forsurvival (p=0.08 by logrank test).

Discussion

Applicants have made considerable efforts to develop gangliosidevaccines that induce high levels of serum antibodies (18, 19) because 1)gangliosides are major cell surface constituents of melanoma cells (14,20-25), 2) human monoclonal antibodies with specificity for gangliosidescan be isolated with relatively high frequency from patients withmelanoma (14, 25-28) and 3) human tumor cells expressing gangliosideantigens can be lysed by anti-ganglioside antibodies in the presence ofhuman complement (13, 29). GM2 ganglioside had been found particularlyimmunogenic in humans, and vaccines containing purified GM2 and BCG asadjuvant have been shown to elicit GM2 antibodies in melanoma patientspretreated with low dose cyclophosphamide (8, 9). The study reportedhere was intended to answer two questions—does vaccination with aGM2/BCG vaccine induce production of GM2 antibodies in a high percentageof melanoma patients and does induction of GM2 antibodies alter thecourse of the disease in patients with Stage III melanoma after completeresection of all known tumor?

Regarding induction of GM2 antibodies, immunization with GM2/BCG wasclearly effective in the majority of patients. Of 58 patients receivingthe GM2/BCG vaccine, 50 produced GM2 antibodies detected in both ELISAand dot blot immune stain assays. As in earlier experience (9), theinduced GM2 antibodies were mostly of the IgM class; IgG antibodies weredetected less frequently. The correlation between GM2 antibodyproduction and a more favorable clinical course (9) was also confirmedhere and expanded in a more homogeneous population of disease-free AJCCStage III melanoma patients. Patients producing GM2 antibody (whethernaturally occurring or vaccine induced) had a significantly longerdisease-free and overall survival than patients who showed no antibodyresponse. Preexisting GM2 antibody (prior to vaccination) was seen insix patients in this series and appeared to be associated with anespecially favorable prognosis. As the incidence of spontaneous GM2antibody production appears to be similar (10%) in the generalpopulation and in melanoma patients (8, 9), their relationship tomelanoma development and growth remains unknown. It is of note that thetiter of preexisting GM2 antibodies did not change over the nine-monthfollow-up period, whereas vaccine-induced GM2 antibodies generallyremained detectable for only 8-12 weeks after immunization. Thisobservation raises the possibility that persistence of antibodyproduction may be important for a favorable clinical outcome and itindicates the need for developing GM2 vaccines that produce long lastingGM2 antibody responses. In contrast to vaccinated patients developingGM2 antibody, the 8 patients who did not produce GM2 antibody afterimmunization with GM2/BCG showed particularly rapid disease progression,recurrence being detected usually before the complete series of fiveimmunizations was administered. Other prognostic factors were not lessfavorable in these patients, nor was their antibody response to BCG lessvigorous than the response in GM2 antibody positive patients, indicatingthat general immunosuppression is not the underlying mechanism.

An association between GM2 antibody production and improved prognosishas also been suggested in studies of the antigen designated OFA-I (31),a cell surface antigenic system expressed by many melanomas. Severallines of evidence have indicated that antibody reactivity against OFA isprimarily directed against GM2 ganglioside (30, 25). Patients withincreased IgM titers against OFA-I, either preexisting or afterimmunization with irradiated melanoma cells, showed prolonged survival(31).

While adding strength to the argument that GM2 antibodies are associatedwith a favorable prognosis in patients with melanoma, this study alsoillustrates the problems that emerge in designing randomized vaccinetrials. Preexisting GM2 antibody production in 5 patients in the controlgroup as opposed to 1 patient in the GM2/BCG group, and lack of a GM2antibody response in 8 vaccinated patients in the GM2/BCG groupcontributed to a blunting of the distinction between the two randomizedtreatment groups to a point that the therapeutic gain in the GM2/BCGgroup was not significant. Clearly, patients with preexisting antibodiesto GM2 need to be excluded or at least stratified in future studies.Equally important is the need to increase the immunogenicity of thevaccine. In this regard, applicants have explored approaches that havebeen successfully pursued in the context of carbohydrate vaccinesagainst bacterial infections (reviewed in 32), including chemicalmodification of gangliosides to yield closely related congeners (33,34), ganglioside conjugation with immunogenic protein carriers (35), anduse of more potent adjuvants (36). The approach applicants have foundmost effective has been to conjugate GM2 with keyhole limpet hemocyanin(KLH) and to administer the GM2-KLH conjugate with QS-21 (a saponinextract from Saponaria Quillaja) as adjuvant. In a pilot study inpatients with melanoma, administration of this vaccine resulted in IgMGM2 antibody titers much higher than those seen after GM2/BCG and, evenmore important, consistent production of GM2 antibodies belonging to theIgG class, thus apparently converting a T-independent antibody responseinto a T-dependent response (37).

An additional approach to improving the clinical efficacy of the GM2/BCGvaccine described here would be incorporation of additional immunogenicmelanoma gangliosides into the vaccine. Experiments in the mouseindicate that conjugation with KLH and use of QS-21 adjuvant alsoaugments the immunogenicity of GD3 (35), a more highly expressedmelanoma ganglioside that has so far shown very low immunogenicity inpatients with melanoma. If these observations can be confirmed in humanstudies, applicants may have the basis for constructing a polyvalentganglioside conjugate vaccine incorporating several major melanomagangliosides, and thus circumventing the heterogeneity of gangliosideexpression seen in human melanomas. Applicants believe that the resultsof the study reported here justify further pursuit of this approach.

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1-43. (canceled)
 44. A composition which comprises: a) a conjugate of(i) a derivative of a GM2 ganglioside, which GM2 ganglioside comprisesan unaltered sphingosine base, wherein the derivative differs from theGM2 ganglioside solely by having an altered sphingosine base whichretains only C1 through C4 from the unaltered sphingosine base of theGM2 ganglioside, and (ii) Keyhole Limpet Hemocyanin, wherein the GM2ganglioside derivative is covalently bound to Keyhole Limpet Hemocyaninby a stable amine bond between the C-4 carbon of the altered sphingosinebase and a nitrogen of an ε-aminolysyl group of Keyhole LimpetHemocyanin; b) a saponin; and c) a pharmaceutically acceptable carrier,wherein the amount of the conjugated GM2 ganglioside derivative is anamount between about 1 μg and about 200 μg, the amount of the saponin isan amount between about 10 μg and about 200 μg, and the GM2:KeyholeLimpet Hemocyanin molar ratio is from 200:1 to 1400:1, the relativeamounts of the conjugate and of the saponin being effective to stimulateor enhance production in a subject of an antibody to the GM2ganglioside.
 45. A method of treating a subject afflicted with a cancerwhich comprises administering to the subject an amount of thecomposition of claim 44 effective to stimulate or enhance production ina subject of an antibody to the GM2 ganglioside and to thereby treat thecancer in the subject.
 46. A method of stimulating or enhancingproduction of an antibody directed to the GM2 ganglioside in a subjectwhich comprises administering to the subject an effective amount of acomposition which comprises: a) a conjugate of (i) a derivative of a GM2ganglioside, which GM2 ganglioside comprises an unaltered sphingosinebase, wherein the derivative differs from the GM2 ganglioside solely byhaving an altered sphingosine base which retains only C1 through C4 fromthe unaltered sphingosine base of the GM2 ganglioside, and (ii) KeyholeLimpet Hemocyanin, wherein the GM2 ganglioside derivative is covalentlybound to Keyhole Limpet Hemocyanin by a stable amine bond between theC-4 carbon of the altered sphingosine base and a nitrogen of anε-aminolysyl group of Keyhole Limpet Hemocyanin; b) a saponin; and c) apharmaceutically acceptable carrier, wherein the amount of theconjugated GM2 ganglioside derivative is an amount between about 1 μgand about 200 μg, the amount of the saponin is an amount between about10 μg and about 200 μg, and the GM2:Keyhole Limpet Hemocyanin molarratio is from 200:1 to 1400:1, the relative amounts of the conjugate andof the saponin being effective to stimulate or enhance production in asubject of an antibody directed to the GM2 ganglioside.
 47. A method oftreating a human subject having cancer which comprises administering tothe subject an effective amount of a composition which comprises: a) aconjugate of (i) a derivative of a GM2 ganglioside which GM2 gangliosidecomprises an unaltered sphingosine base, wherein the derivative differsfrom the GM2 ganglioside solely by having an altered sphingosine basewhich retains only C1 through C4 from the unaltered sphingosine base ofthe GM2 ganglioside, and (ii) Keyhole Limpet Hemocyanin, wherein the GM2ganglioside derivative is covalently bound to Keyhole Limpet Hemocyaninby a stable amine bond between the C-4 carbon of the altered sphingosinebase and a nitrogen of an ε-aminolysyl group of Keyhole LimpetHemocyanin; and b) a saponin; and c) a pharmaceutically acceptablecarrier, wherein the amount of the conjugated GM2 ganglioside derivativeis an amount between about 1 μg and about 200 μg, the amount of saponinis an amount between about 10 μg and about 200 μg, the GM2derivative:Keyhole Limpet Hemocyanin molar ratio is from 200:1 to1400:1, the relative amounts of the conjugate and of the saponin beingeffective to stimulate or enhance production in a subject of an antibodyto the GM2 ganglioside and thereby treat the subject.